JP2014514331A - Tolerogenic synthetic nanocarriers - Google Patents

Abstract

  The present invention relates to a composition comprising a synthetic nanocarrier and an immunosuppressive agent that at least partially provides an immunosuppressive effect. Such compositions can further comprise an antigen and provide an antigen-specific tolerogenic immune response.

Description

RELATED APPLICATIONS This application is filed under United States Patent Law Section 119, US Provisional Patent Application No. 61 / 480,946, filed April 29, 2011, and filed July 29, 2011. 61 / 513,514, 61 / 631,147 filed on September 6, 2011, 61 / 531,153 filed September 6, 2011, September 6, 2011 61 / 531,164 filed on the same day, 61 / 531,168 filed on September 6, 2011, 61 / 531,175 filed on September 6, 2011 No. 61 / 531,180 filed on September 6, 2011, 61 / 631,194 filed on September 6, 2011, filed September 6, 2011 No. 61 / 531,204, filed on September 6, 2011 No. 61 / 531,209 and No. 61 / 531,215 filed on September 6, 2011, and the entire contents of each of these provisional patent applications are Which is incorporated herein by reference.

  The present invention relates, at least in part, to a composition comprising a synthetic nanocarrier and an immunosuppressive agent that can provide an antigen-specific immunosuppression, preferably a tolerogenic immune response.

  Conventional approaches for generating immunosuppression associated with undesirable immune responses are based on widely acting immunosuppressive agents. In addition, immunosuppressant therapy is generally a lifelong plan to maintain immunosuppression. Unfortunately, the use of widely acting immunosuppressive agents carries the risk of serious side effects such as tumors, infections, nephrotoxicity and metabolic abnormalities. Therefore, a novel immunosuppressant therapy would be beneficial.

  In one aspect, a composition comprising (i) a first population of synthetic nanocarriers coupled to an immunosuppressive agent and (ii) an APC-presentable antigen is provided. In one embodiment, the immunosuppressive agent in the composition is an amount effective to generate a tolerogenic immune response against the APC presentable antigen. In one embodiment, the APC presentable antigen is bound to a first group of synthetic nanocarriers of synthetic nanocarriers. In another embodiment, the APC presentable antigen is bound to a second population of synthetic nanocarriers of synthetic nanocarriers.

  In one embodiment, the first population and the second population are the same.

  In one embodiment, the average particle size distribution obtained using dynamic light scattering of the first and / or second population of synthetic nanocarriers is a diameter greater than 100 nm. In another embodiment, the diameter is greater than 150 nm. In another embodiment, the diameter is greater than 200 nm. In another embodiment, the diameter is greater than 250 nm. In another embodiment, the diameter is greater than 300 nm.

  In one embodiment, the immunosuppressive agent is a statin, mTOR inhibitor, TGF-β signaling agent, corticosteroid, mitochondrial function inhibitor, P38 inhibitor, NF-κβ inhibitor, adenosine receptor agonist, prostaglandin. Gin E2 agonist, phosphodiesterase 4 inhibitor, HDAC inhibitor or proteasome inhibitor. In another embodiment, the mTOR inhibitor is rapamycin or an analog thereof.

  In one embodiment, the APC presentable antigen comprises MHC class I restricted and / or MHC class II restricted and / or B cell epitopes. In another embodiment, the APC presentable antigen is a protein. In another embodiment, the APC presentable antigen is a polypeptide or peptide comprising MHC class I restricted and / or MHC class II restricted and / or B cell epitopes. In another embodiment, the APC presentable antigen is a lipid that binds to CD1d. In one embodiment, the APC presentable antigen is a therapeutic protein or portion thereof, autoantigen or allergen, or is associated with autoimmune disease, inflammatory disease, allergy, organ or tissue rejection or graft-versus-host disease To do.

  In one embodiment, the load of immunosuppressive agent and / or APC presentable antigen is 0.0001% to 50% (weight / weight), averaged over the entire population of the first and / or second synthetic nanocarriers. It is. In another embodiment, the immunosuppressive agent and / or APC presentable antigen loading is 0.1% to 10% (weight / weight) on average over the entire population of the first and / or second synthetic nanocarriers. ).

  In one embodiment, the synthetic nanocarriers of the first and / or second population of synthetic nanocarriers are lipid nanoparticles, polymer nanoparticles, metal nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, Includes virus-like particles or peptide or protein particles.

  In one embodiment, where the first and / or second population of synthetic nanocarriers of synthetic nanocarriers comprise polymer nanoparticles, the polymer nanoparticles comprise a polymer that is a non-methoxy terminated pluronic polymer. In another embodiment, the polymer nanoparticles comprise polyester, polyester bonded to a polyether, polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline or polyethyleneimine. In another embodiment, the polyester comprises poly (lactic acid), poly (glycolic acid), poly (lactic acid-co-glycolic acid) or polycaprolactone. In another embodiment, the polymer nanoparticles comprise a polyester bonded to a polyester and polyether. In another embodiment, the polyether comprises polyethylene glycol or polypropylene glycol.

  In another embodiment, the aspect ratio of the synthetic nanocarriers of the first and / or second population of synthetic nanocarriers is 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: More than 3, 1: 5, 1: 7 or 1:10.

  In another embodiment, the composition further comprises a pharmaceutically acceptable excipient.

  In another aspect, dosage forms comprising any of the provided compositions are provided.

  In another aspect, a method is provided that comprises administering to a subject any of the compositions or dosage forms provided herein. In one embodiment, the subject is in need of antigen-specific tolerance. In another embodiment, the subject has an autoimmune disease, inflammatory disease, allergy, graft-versus-host disease, organ or tissue rejection, or has undergone or will undergo a transplant. In another embodiment, the subject has been administered, is being administered, or will be administered a therapeutic protein that has, has, or is expected to have an undesirable immune response.

  In another embodiment, any of the provided compositions or dosage forms is or is administered in an amount effective to produce a tolerogenic immune response specific for an APC-presentable antigen. obtain. In another embodiment, the dosage form is administered to a subject according to a protocol already known to produce a tolerogenic immune response specific for APC that can be presented in one or more subjects.

  In another embodiment of any of the provided compositions or dosage forms, the composition may produce a desired immune response or reduce an undesirable immune response in a subject, for example.

  In one embodiment, the method further comprises providing or identifying a subject. In another embodiment, the method further comprises assessing the generation of a tolerogenic immune response specific for the APC presentable antigen in the subject.

  In one embodiment, the dosage form is administered by intravenous, transmucosal, intraperitoneal, oral, subcutaneous, transpulmonary, intranasal, intradermal or intramuscular administration. In another embodiment, administration is by inhalation or intravenous, subcutaneous or transmucosal administration.

  In another embodiment, any of the provided compositions or dosage forms can be for use in therapy or prevention.

  In another aspect, any of the provided compositions or dosage forms have undergone, or are scheduled to undergo, transplantation, or have caused, or are expected to cause, an undesirable immune response. The therapeutic protein is administered, may be for use in the treatment or prevention of a subject being administered or will be administered.

  In another aspect, any of the provided compositions or dosage forms can be for use in any of the provided methods.

  In another embodiment, any of the provided compositions or dosage forms can be for use in a method of inducing a tolerogenic immune response.

  In another aspect, any of the provided compositions or dosage forms is a method of preventing or treating autoimmune disease, inflammatory disease, allergy, organ or tissue rejection, graft-versus-host disease or undesirable immune response. Can be for use in.

  In another embodiment, any of the provided compositions or dosage forms can be for use in a method of treatment or prevention that includes administration by any of the routes provided herein.

  In another aspect, there is provided use of any of the compositions or dosage forms provided for the manufacture of a medicament for use in any of the provided methods.

  In one embodiment of any of the compositions and methods provided herein, the antigen is a peptide. Such antigens, in certain embodiments, include at least one epitope described elsewhere herein, but may also include additional amino acids adjacent to one or both ends of the epitope. In embodiments, the antigen comprises the whole antigen protein. These antigens can be bound to synthetic nanocarriers.

A representative example of flow cytometric analysis of Treg cells is provided. Figure 5 shows antigen-specific induction of FoxP3 ⁺ in CD4 ⁺ CD25high Treg cells by tDC treated with nanocarrier-encapsulated rapamycin and free ovalbumin (323-339). FIG. 6 shows antigen-specific induction of FoxP3 ⁺ in CD4 ⁺ CD25high Treg cells. Figure 3 shows the effect on the number of antigen-specific effector T cells and the percentage of FoxP3 + cells by a synthetic nanocarrier of the present invention containing an immunosuppressant (rapamycin or simvastatin). FIG. 5 shows a reduction in the number of popliteal lymph node cells by the synthetic nanocarriers of the present invention containing an immunosuppressant (rapamycin or simvastatin). FIG. 9 shows the reduction of CD69, a marker of T cell activation, by synthetic nanocarrier delivery of the immunosuppressant rapamycin.

  Before describing the present invention in detail, it is to be understood that the present invention is not limited to the specifically exemplified materials or process parameters and may, of course, vary. The terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to limit the use of other terminology that describes the invention. It is also understood that there is no.

  All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes, whether as described above or below.

  As used in this specification and the appended claims, the singular forms ("a", "an", and "the") refer to the plural unless the context clearly dictates otherwise. Includes subject. For example, reference to “a polymer” includes a mixture of two or more such molecules or a mixture of single polymer species of different molecular weights, and a reference to “a synthetic nanocarrier”. Includes two or more such synthetic nanocarriers or a mixture of a plurality of such synthetic nanocarriers, and a reference to “a DNA molecule” refers to two or more such DNA molecules or Including a mixture of a plurality of such DNA molecules, references to “an immunosuppressant” include a mixture of two or more such materials or a plurality of immunosuppressant molecules, and the like.

  As used herein, the term “comprise” or variations thereof such as “comprises” or “comprising” may be any described integer (eg, feature, element , Characteristics, properties, methods / process steps or restrictions) or integers (eg, features, elements, characteristics, properties, methods / process steps or restrictions) should be read to indicate the inclusion of the group, but any other Does not exclude integers or groups of integers. Thus, as used herein, the term “comprising” is inclusive and does not exclude additional undescribed integers or method / process steps.

  In any embodiment of the compositions and methods provided herein, “comprising” is replaced with “consisting essentially of” or “consisting of”. May be. The phrase “consisting essentially of” herein requires a specified integer or process and an integer or process that does not substantially affect the nature or function of the claimed invention. Used to do. As used herein, the term “consisting” refers to an integer (eg, feature, element, property, property, method / process step or limit) or integer (eg, feature, element) described. , Properties, properties, methods / process steps or restrictions) are used to indicate the presence of only a group.

A. Introduction As noted above, current conventional immunosuppressive agents work widely and generally result in an overall systemic down-regulation of the immune system. The compositions and methods provided herein allow for a more targeted immune effect, for example by allowing targeted delivery to a subject immune cell. Thus, the present compositions and methods can provide more targeted immunosuppression. As shown in the Examples, the composition of the present invention was used to generate regulatory cells and successfully reduce T cell activation as evidenced by a decrease in CD69.

  The inventors have unexpectedly and unexpectedly discovered that the above problems and limitations can be overcome by practicing the invention disclosed herein. In particular, we have unexpectedly discovered that it is possible to provide compositions and related methods that provide immunosuppression, in certain embodiments, antigen-specific tolerogenic effects. did. The compositions described herein are compositions comprising 1) a population of synthetic nanocarriers conjugated with an immunosuppressant and 2) an antigen presenting cell (APC) presentable antigen. Efficient uptake of such compositions by APC favors immune responses, such as regulatory T (Treg) cell generation specific for APC presentable antigens, for immunosuppressive effects (eg tolerogenic effects). An in vivo and in vitro convenient method of changing.

  In one embodiment, the APC presentable antigen is bound to the same synthetic nanocarrier to which the immunosuppressive agent is bound. In another embodiment, APC presentable antigens are bound to different synthetic nanocarriers. In yet another embodiment, the APC presentable antigen is not bound to a synthetic nanocarrier. In another embodiment, the immunosuppressive agent in the composition is an amount effective to generate a tolerogenic immune response against an APC presentable antigen.

  In yet another embodiment, the immunosuppressant loading is 0.0001% to 50% (weight / weight) on average for the entire population of synthetic nanocarriers. In a further embodiment, the APC presentable antigen loading is 0.0001% to 50% (w / w) on average over the entire population of synthetic nanocarriers. Preferably, in certain embodiments, the loading of the immunosuppressant is 0.1% to 10% (weight / weight) on average over the entire population of synthetic nanocarriers and / or the loading of APC presentable antigen Is 0.1% to 10% (weight / weight) on average for the entire population of synthetic nanocarriers.

  In another aspect, dosage forms of any of the compositions herein are provided. Such dosage forms can be administered to a subject as needed (eg, requiring antigen-specific tolerance). In one embodiment, the subject is a subject suffering from or at risk of suffering from an autoimmune disease, inflammatory disease, organ or tissue rejection, graft-versus-host disease or allergy. In another embodiment, the subject is a subject who has or will be transplanted. In yet another embodiment, the subject is a subject who has been or will be treated with a therapeutic agent that stimulates an undesirable immune response.

  The invention will now be described in more detail below.

B. Definitions “Administering” or “administration” means providing material to a subject in a pharmacologically useful manner.

  An “allergen” is any substance that can cause an undesirable (eg, type 1 hypersensitivity) immune response (ie, an allergic response or reaction) in a subject. Allergens include, but are not limited to, plant allergens (eg, pollen, ragweed allergen), insect allergens, insect sting allergens (eg, bee sting allergen), animal allergens (eg, animal scales or feline Fel d 1 antigen) Pet allergens, etc.), latex allergens, mold allergens, fungal allergens, cosmetic allergens, drug allergens, food allergens, dust, insect venoms, viruses, bacteria and the like. Food allergens include, but are not limited to, milk allergens, egg allergens, nut allergens (eg, peanuts or nuts allergens (eg, walnuts, cashews)), fish allergens, crustacean allergens, soy allergens, legumes Allergens, seed allergens and wheat allergens. Insect stings allergens include bee stings, wasp stings, hornet stings, yellow jacket stings, and related allergens. Insect allergens also include mite allergens (eg, Der P1 antigen) and cockroach allergens. Drug allergens include allergens that are or are related to antibiotics, NSAIDs, anesthetics, and the like. Pollen allergens include grass allergens, tree allergens, weed allergens, flower allergens and the like. A subject who develops or is at risk of developing an undesirable immune response to any of the allergens provided herein can be treated with any of the compositions and methods provided herein. Subjects that can be treated with any of the provided compositions and methods also include subjects who have or are at risk of having an allergy to any of the provided allergens.

  An “allergy”, also referred to herein as an “allergic condition”, is any condition in which there is an undesirable (eg, type 1 hypersensitivity) immune response (ie, allergic response or reaction) to a substance. Such substances are referred to herein as allergens. Allergic or allergic conditions include, but are not limited to, allergic asthma, hay fever, urticaria, eczema, plant allergy, bee sting allergy, pet allergy, latex allergy, mold allergy, cosmetic allergy, food allergy, allergy Include rhinitis or nasal cold, local allergic reactions, anaphylaxis, atopic dermatitis, hypersensitivity reactions and other allergic conditions. An allergic reaction can be the result of an immune response to some allergen. In certain embodiments, the allergy is a food allergy. Food allergies include, but are not limited to, milk allergies, egg allergies, nut allergies, fish allergies, crustacean allergies, soybean allergies or wheat allergies.

  An “effective amount” in the context of a composition or dosage form for administration to a subject produces one or more desirable immune responses in the subject (eg, generation of a tolerogenic immune response), or Refers to the amount of the dosage form. Thus, in certain embodiments, an effective amount is any amount of a composition provided herein that produces one or more of these desired immune responses. This amount can be for in vitro or in vivo purposes. For in vivo purposes, the amount can be the amount that a clinician would consider to have a clinical effect on a subject in need of antigen-specific tolerance. Such subjects include subjects suffering from or at risk of suffering from inflammatory diseases, autoimmune diseases, allergies, organ or tissue rejection or graft-versus-host disease. Such subjects also include subjects who have or will be transplanted. Such subjects further include subjects that have, have, or are expected to have an undesirable immune response to the therapeutic protein.

  While an effective amount can only be involved in reducing the level of an undesirable immune response, in certain embodiments, an effective amount is responsible for completely preventing an undesirable immune response. An effective amount may also be involved in delaying the development of undesirable immune responses. An effective amount can also be an amount of a composition provided herein that provides a desired therapeutic endpoint or desired therapeutic outcome. An effective amount preferably results in the subject's tolerogenic immune response to the antigen. Any of the above outcomes can be monitored by routine methods.

  In certain embodiments of any of the provided compositions and methods, the effective amount is such that the desired immune response is at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 3 months in the subject. For at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, at least 5 years or more. In other embodiments of any of the provided compositions and methods, an effective amount is a measurable desired immune response, such as at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 3 An immune response (eg, against a particular antigen) over a period of at least 4 months, at least 5 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, at least 5 years, or more An amount that results in a measurable decrease in.

  The effective amount will, of course, be the specific subject being treated; the severity of the condition, disease or disorder; individual patient parameters including age, health status, size and weight; duration of treatment; combination therapy (if any) ) Nature; it will depend on the specific route of administration and similar factors within the knowledge and expertise of the healthcare professional. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred that the maximum dose be used, that is, the highest dose that is safe according to reasonable medical judgment. However, it will be appreciated by those skilled in the art that a patient may require a lower dose or tolerated dose for medical reasons, psychological reasons or virtually any other reason.

In general, the dose of immunosuppressive agent and / or antigen in the composition of the present invention may range from about 10 μg / kg to about 100,000 μg / kg. In certain embodiments, the dosage can range from about 0.1 mg / kg to about 100 mg / kg. In still other embodiments, the dosage is from about 0.1 mg / kg to about 25 mg / kg, from about 25 mg / kg to about 50 mg / kg, from about 50 mg / kg to about 75 mg / kg, or from about 75 mg / kg to about 100 mg. / Kg range. Alternatively, the dosage can be administered based on the number of synthetic nanocarriers from which the desired amount of immunosuppressant and / or antigen is obtained. For example, useful doses include more than 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ or 10 ¹⁰ synthetic nanocarriers per dose. Other examples of useful doses are about 1 × 10 ⁶ to about 1 × 10 ¹⁰ , about 1 × 10 ⁷ to about 1 × 10 ⁹ or about 1 × 10 ⁸ to about 1 × 10 ⁹ per dose. Contains synthetic nanocarriers.

  “Antigen” means a B cell antigen or a T cell antigen. “Antigen type” means molecules that share the same or substantially the same antigenic characteristics. In certain embodiments, the antigen can be a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, polysaccharide, or contained in or expressed in a cell. In certain embodiments, such as when the antigen is not clearly defined or characterized, the antigen may be contained in a cell or tissue specimen, cell debris, cell exosomes, conditioned media, and the like. The antigen may be combined with the synthetic nanocarrier in the same form that causes an undesirable immune response to which the subject is exposed, but may also be a fragment or derivative thereof. However, in the case of fragments or derivatives, the desired immune response to the form to which such subjects are exposed is a favorable result with the provided compositions and methods.

  “Antigen-specific” refers to any immune response that results from the presence of an antigen, or portion thereof, or that produces a molecule that specifically recognizes or binds the antigen. For example, if the immune response is antigen-specific antibody production, antibodies that specifically bind the antigen are produced. As another example, if the immune response is antigen-specific B cell or T cell proliferation and / or activity, the proliferation and / or activity is recognized by the B cell or the like, or only a portion thereof, or an MHC molecule Obtained from the recognition of the antigen complexed with or part thereof.

  As used herein, an “associated antigen” with a disease, disorder or condition is an undesirable immune response to, or associated with, the disease, disorder or condition; the disease, disorder or condition (or its symptoms or And / or antigens that can produce an undesirable immune response that is a symptom, consequence or action of a disease, disorder or condition. Preferably, in certain embodiments, the use of an antigen associated with a disease, disorder or condition, etc. in the compositions and methods provided herein provides that the antigen and / or the antigen is expressed on or in the cell. Produces a tolerogenic immune response to The antigen may be in the same form as expressed in a subject afflicted with a disease, disorder or condition, but may also be a fragment or derivative thereof. However, in the case of fragments or derivatives, the desired immune response to the form expressed in such subjects is a favorable result with the provided compositions and methods. Antigens associated with a disease, disorder or condition, in certain embodiments, include MHC class I restricted epitopes and / or MHC class II restricted epitopes and / or B cell epitopes and / or bind to the CD1d complex. , Including lipids that form the CD1d complex.

  In one embodiment, the antigen is an antigen associated with inflammatory disease, autoimmune disease, organ or tissue rejection or graft-versus-host disease. Such antigens include myelin basic protein, collagen (eg, type 11 collagen), human cartilage gp39, chromogranin A, gp130-RAPS, proteolipid protein, fibrillarin, nucleoprotein, nucleolus protein (eg, small body protein) Nucleolar protein), thyroid stimulating factor receptor, histone, glycoprotein gp 70, ribosomal protein, pyruvate dehydrogenase dihydrolipoamide acetyltransferase, hair follicle antigen, human tropomyosin isoform 5, mitochondrial protein, pancreatic β cell protein, myelin Self-antigens such as oligodendrocyte glycoproteins, insulin, glutamate decarboxylase (GAD), gluten, and fragments or derivatives thereof. Other autoantigens are shown in Table 1 below.

  Antigens also include antigens associated with organ or tissue rejection. Examples of such antigens include, but are not limited to, antigens from other cells, such as antigens from allogeneic cells, such as antigens from allogeneic cell extracts and endothelial cell antigens. It is done.

  Antigens also include antigens associated with allergies. Such antigens include allergens described elsewhere herein.

  Antigens also include antigens associated with implantable grafts. Such antigens are associated with an undesired immune response in a transplantable graft or a transplantable graft recipient that is generated as a result of introduction of the transplantable graft into the recipient, and the immune system May be presented for recognition by the cell and produce an undesirable immune response. Transplant antigens include those associated with organ or tissue rejection or graft-versus-host disease. Transplant antigens can be obtained from or derived from information related to cells of biomaterial or transplantable grafts. Transplant antigens generally include proteins, polypeptides, peptides, lipoproteins, glycolipids, polynucleotides, or are contained in or expressed in cells. Information related to an implantable implant is any information about the implantable implant that can be used to obtain or remove a transplant antigen. Such information can be found, for example, in cells of an implantable graft, such as antigenic sequence information, type or class and / or restriction of their MHC class I, MHC class II or B cell presentation. Or contains information about antigens that may be expected to be present on the cell. Such information includes the type of implant that is implantable (eg, autograft, allograft, xenograft), the molecular and cellular composition of the graft, the body site or graft from which the graft is derived. It may also include information about the body part to be (eg, whole or partial organ, skin, bone, nerve, tendon, neuron, blood vessel, fat, cornea, etc.).

  Antigens also include antigens associated with therapeutic proteins that can be presented for recognition by cells of the immune system and can generate an undesirable immune response against the therapeutic protein. Therapeutic protein antigens generally include proteins, polypeptides, peptides, lipoproteins, or are contained or expressed in, on or on a cell.

  The antigen can be a well-defined or characterized antigen. However, in certain embodiments, the antigen is not well defined or characterized. Thus, the antigen is contained in a cell or tissue specimen, cell debris, cell exosome or conditioned medium, and in certain embodiments also includes an antigen that can be delivered in such a form.

By “APC presentable antigen” is meant an antigen that can be presented for recognition by cells of the immune system, presented by antigen presenting cells including, but not limited to, dendritic cells, B cells or macrophages. APC presentable antigens can be presented for recognition by, for example, T cells. Such antigens are recognized by the immune response in T cells by binding to a class I or class II major histocompatibility complex molecule (MHC) or by presenting an antigen or portion thereof bound to a CD1d molecule. Get and cause it. CD1d is an antigen presenting molecule that binds self and foreign lipids and glycolipids and is often found in antigen presenting cells. CD1d is also found in non-hematopoietic cells such as hepatocytes. CD1d usually contains a hydrophobic groove that binds hydrophobic lipids for presentation to iNKT cells. Preferably, one or more tolerogenic immune responses specific for APC presentable antigens are generated by the compositions provided herein. Such an immune response can be influenced by stimulation, production, induction or mobilization of regulatory cells such as, for example, CD4 ⁺ Treg cells and / or CD8 ⁺ Treg cells.

  APC presentable antigens generally include peptides, polypeptides, whole proteins or whole cell lysates. In one embodiment, the APC presentable antigen comprises an MHC class I restricted epitope. In another embodiment, the APC presentable antigen comprises an MHC class II restricted epitope. In another embodiment, the APC presentable antigen comprises a B cell epitope. However, in another embodiment, the APC presentable antigen is a lipid that binds to or forms the CD1d complex.

  In a further embodiment, the APC presentable antigen in the composition of the invention is provided in the form of a nucleic acid encoding a peptide, polypeptide or protein. The nucleic acid can be DNA or RNA such as mRNA. In embodiments, the compositions of the invention include complement, such as the full-length complement of any of the nucleic acids provided herein, or altered tissue (due to altered genetic code). In embodiments, the nucleic acid is an expression vector that can be transcribed when transfected into a cell line. In embodiments, the expression vector may include, in particular, a plasmid, retrovirus, or adenovirus.

  In one embodiment, the antigen is associated with a disease, disorder or condition described herein and may be combined with an immunosuppressive agent to provide a tolerogenic immune response specific for the disease, disorder or condition.

  “Evaluation of immune response” refers to any measurement or determination, such as the level, presence or absence, decrease, increase, etc. of an immune response in vitro or in vivo. Such measurements or determinations can be made on one or more samples obtained from a subject. Such an assessment can be performed using either the methods provided herein or other methods known in the art.

  A “risk” subject is one who has the potential for suffering from a disease, disorder or condition provided herein, or a subject that is considered by a healthcare professional, or an undesirable immune response provided herein. A subject considered by a medical professional to be able to wake up.

  An “autoimmune disease” is any disease in which the immune system produces an undesirable immune response against itself (eg, one or more self-antigens). In certain embodiments, the autoimmune disease comprises abnormal destruction of the body's cells as part of a self-targeted immune response. In certain embodiments, the self-destruction is manifested in dysfunction of an organ, such as the colon or pancreas. Examples of autoimmune diseases are described elsewhere in this specification. Additional autoimmune diseases are known to those skilled in the art and the invention is not limited in this regard.

  “Average” as used herein refers to the arithmetic average unless otherwise specified.

  “B cell antigen” means any antigen that elicits an immune response in a B cell (eg, an antigen that is specifically recognized by a B cell or a receptor thereon). In certain embodiments, an antigen that is a T cell antigen is also a B cell antigen. In other embodiments, the T cell antigen is also not a B cell antigen. B cell antigens include, but are not limited to, proteins, peptides, small molecules, and carbohydrates. In certain embodiments, the B cell antigen comprises a non-protein antigen (ie, does not comprise a protein or peptide antigen). In certain embodiments, the B cell antigen comprises an autoantigen. In other embodiments, the B cell antigen is obtained from or derived from an allergen, autoantigen, therapeutic protein, or implantable graft.

  “Simultaneously” means that two or more substances are administered to a subject that are temporally correlated, preferably sufficiently correlated to provide modulation of the immune response. In embodiments, co-administration can be performed by administration of two or more substances in the same dosage form. In other embodiments, co-administration is the administration of two or more substances in different dosage forms, provided that they are within a defined period, preferably within one month, more preferably within one week, even more preferably within one day, Even more preferably, administration within 1 hour may be included.

  “Coupled” or “bonded” or “bonded” (such as) means that substances (eg, moieties) are chemically bonded to each other. In certain embodiments, the binding is covalent, meaning that the binding occurs in a situation where there is a covalent bond between the two substances. In non-covalent embodiments, non-covalent bonds include charge interactions, affinity interactions, metal coordination, physisorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, There are non-covalent interactions, including but not limited to van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-to-dipole interactions, and / or combinations thereof. In embodiments, the encapsulation is in the form of a bond.

  “Dosage form” means a pharmacologically and / or immunologically active material in a vehicle, carrier, vehicle, or device suitable for administration to a subject.

  “Encapsulating” means placing at least a portion of the material in a synthetic nanocarrier. In certain embodiments, the material is fully encased in the synthetic nanocarrier. In other embodiments, most or all of the encapsulated material is not exposed to the local environment outside the synthetic nanocarrier. In other embodiments, 50%, 40%, 30%, 20%, 10% or 5% (weight / weight) or less is exposed to the local environment. Encapsulation is different from absorption, which places most or all of the material on the surface of the synthetic nanocarrier and leaves the material exposed to the local environment outside the synthetic nanocarrier.

  An “epitope”, also known as an antigenic determinant, is a portion of an antigen that is recognized by the immune system, in particular, for example, by antibodies, B cells, or T cells. As used herein, an “MHC class I restricted epitope” is an epitope presented to immune cells by MHC class I molecules found in nucleated cells. “MHC class II restricted epitopes” are MHC found in antigen presenting cells (APCs), eg, in professional antigen presenting immune cells such as macrophages, B cells, and dendritic cells, or in non-hematopoietic cells such as hepatocytes. An epitope presented to immune cells by class II molecules. A “B cell epitope” is a molecular structure recognized by an antibody or B cell. In certain embodiments, the epitope itself is an antigen.

  Several epitopes are known to those of skill in the art, and suitable exemplary epitopes according to some aspects of the present invention include, but are not limited to, those listed in the Immuno Epitope Database ( www.Immuneepitope.org, Vita R, Zarebski L, Greenbaum JA, Emami H, Hoof I, Salimi N, Damle R, Sette A, Peters B. Thee. Database issue): D854-62; all of these contents as well as all database entries of IEDB version 2.4 (August 2011), specifically , All epitopes disclosed therein, is incorporated herein by reference). Epitopes are described in published algorithms such as Wang P, Sidney J, Kim Y, Sette A, Lund O, Nielsen M, Peters B. et al. 2010. peptide binding predictions for HLA DR, DP and DQ molecules. BMC Bioinformatics 2010, 11: 568; Wang P, Sidney J, Dow C, Mothe B, Sette A, Peters B. et al. 2008. A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach. PLoS Compute Biol. 4 (4): e1000048; Nielsen M, Lund O .; 2009. NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction. BMC Bioinformatics. 10: 296; Nielsen M, Lundegaard C, Lund O .; 2007. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method. BMC Bioinformatics. 8: 238; Bui HH, Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton KA, Thethe BR, Chisari FV, Watkins DI, Sette A .; 2005. Immunogenetics. 57: 304-314; Sturnio T, Bono E, Ding J, Raddrizzani L, Tuereci O, Sahin U, Braxenthaler M, Gallazzi F, Protti MP, Sinigalia F, Hammer J. 1999. Generation of tissue-specific and promiscuous HLA ligands and using DNA microarrays and virtual HLA class II materials. Nat Biotechnol. 17 (6): 555-561; Nielsen M, Lundegaard C, Worn P, Lauemoller SL, Lamberth K, Busus S, Brunak S, Lund O .; 2003. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci 12: 1007-1017; Bui HH, Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton KA, Mothe BR, Chisari FV, Watkins DI, Sette A. 2005. Automated generation and evaluation of specific MHC binding predictive tools: ARB matrix applications. Immunogenetics 57: 304-314; Peters B, Sette A. et al. 2005. Generating quantitative models describing the sequence specificity of biologic processes with the stabilized matrix method. BMC Bioinformatics 6: 132; Chou PY, Fasman GD. 1978. Prediction of the secondary structure of proteins of the amino acid sequence. Adv Enzymol Relat Areas Mol Biol 47: 45-148; Emini EA, Hughes JV, Perlow DS, Boger J. et al. 1985. Induction of hepatitis A virus-neutralizing activity by a virus-specific synthetic peptide. J Virol 55: 836-839; Karplus PA, Schulz GE. 1985. Prediction of chain flexibility in proteins. Naturewissenschaften 72: 212-213; Kolaskar AS, Tongaonkar PC. 1990. A semi-empirical method for prediction of antigenic detergents on protein antigens. FEBS Lett 276: 172-174; Parker JM, Guo D, Hodges RS. 1986. New hydrophility scale derived from high-performance liquid slidable retention-xenided retentied re-stressed data. Biochemistry 25: 5425-5432; Larsen JE, Lund O, Nielsen M .; 2006. Improved method for predicting linear B-cell epitopes. Immunores Res 2: 2; Ponomarenko JV, Bourne PE. 2007. Antibody-protein interactions: benchmark datasets and prediction tools evaluation. BMC Struct Biol 7:64; Haste Andersen P, Nielsen M, Lund O .; 2006. Prediction of residences in discontinous B-cell epitopes using protein 3D structures. Protein Sci 15: 2558-2567; Ponomarenko JV, Bui H, Li W, Fusseder N, Bourne PE, Sette A, Peters B. et al. 2008. EliPro: a new structure-based tool for the prediction of antibacterial epitopes. BMC Bioinformatics 9: 514; Nielsen M, Lundegaard C, Blicher T, Peters B, Sette A, Justen S, Busus S, and Lund O. 2008. PLoS Compute Biol. 4 (7) e1000107. Quantitative predictions of peptide binding to any HLA-DR molecule of known sequence: The entire contents of each of these can be identified for the identification of epitopes; And incorporated herein by reference.

  Other examples of epitopes that can be bound to the synthetic nanocarriers provided herein include any of the MHC class I restricted, MHC class II restricted and B cell epitopes shown as SEQ ID NOs: 1-943. While not wishing to be bound by any particular theory, MHC class I restricted epitopes include those set forth in SEQ ID NOs: 1-186, and MHC class II restricted epitopes include SEQ ID NO: 187- B cell epitopes include those described in SEQ ID NOs: 538-943. These epitopes include MHC class I restricted autoantigens of allergens, MHC class II restricted epitopes and B cell epitopes of self antigens and allergens.

  “Generation” can be directly on its own or indirectly, including but not limited to an irrelevant third party acting on trust in a person's behavior, such as an immune response (eg, a tolerogenic immune response) Means to cause action.

  “Identification” is any action or series of actions that allow a clinician to recognize a subject as a subject that would benefit from the methods and compositions provided herein. Preferably, the subject identified is a subject in need of the tolerogenic immune response provided herein. The action or series of actions may be performed directly on its own or indirectly, such as, but not limited to, an irrelevant third party who takes action by trusting in human speech.

  “Immunosuppressant” means a compound that causes APC to have an immunosuppressive effect (eg, a tolerogenic effect). An immunosuppressive effect generally refers to the production or expression of cytokines or other factors by APC that reduce, inhibit or prevent an unwanted immune response or promote a desired immune response. If APC provides an immunosuppressive effect on immune cells that recognize the antigen presented by APC, the immunosuppressive effect is considered to be specific for the presented antigen. Such an effect is also referred to herein as a tolerogenic effect. Without being bound by any particular theory, immunosuppression is preferably immunosuppression delivered to the APC in the presence of an antigen (eg, an administered antigen or an antigen already present in the body). It is thought to be the result of the agent. Thus, an immunosuppressive agent includes a compound that confers a tolerogenic immune response to an antigen, which may or may not be provided in the same or different compositions. In one embodiment, the immunosuppressive agent is one that causes APC to promote a regulatory phenotype in one or more immune effector cells. For example, the regulatory phenotype can be characterized by the production, induction, stimulation or mobilization of regulatory immune cells. This may be the result of the conversion of CD4 + T cells (eg, CD4 + CD25highFoxP3 + Treg cells) to a regulatory phenotype. This may also be the result of the induction of FoxP3 in other immune cells such as CD8 + T cells, macrophages and iNKT cells. In one embodiment, an immunosuppressive agent is one that affects the APC response after it has processed the antigen. In another embodiment, the immunosuppressive agent does not interfere with antigen processing. In further embodiments, the immunosuppressive agent is not an apoptotic signaling molecule. In another embodiment, the immunosuppressive agent is not a phospholipid.

  Immunosuppressants include, but are not limited to, statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibition such as trichostatin A Agents; corticosteroids; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-κβ inhibitors such as 6Bio, dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists; prostagland such as misoprostol Gin E2 agonist (PGE2); Phosphodiesterase inhibitors such as phosphodiesterase 4 inhibitor (PDE4) such as rolipram; Proteasome inhibitors; Kinase inhibitors; G protein coupled receptor agonists; G protein coupled receptor antagonists; Glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibition Phosphatase inhibitors; PI3KB inhibitors such as TGX-221; autophagy inhibitors such as 3-methyladenine; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and P2X receptor blockers Of oxidized ATP. Examples of immunosuppressants include cyclosporine such as IDO, vitamin D3, cyclosporin A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6- TG), FK506, sangliferin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol and triptolide. In embodiments, the immunosuppressive agent can include any of the agents provided herein.

  An immunosuppressive agent can be a compound that directly provides an immunosuppressive (eg, tolerogenic) effect on APC or an immunosuppressive (eg, tolerogenic) effect indirectly (ie, treated in some way after administration). Can be given compound). Accordingly, immunosuppressive agents include any prodrug form of the compounds provided herein.

  Immunosuppressive agents also include nucleic acids encoding the peptides, polypeptides or proteins provided herein that provide an immunosuppressive (eg, tolerogenic) immune response. Accordingly, in embodiments, an immunosuppressive agent is a nucleic acid that encodes a peptide, polypeptide or protein that provides an immunosuppressive (eg, tolerogenic) immune response and is a nucleic acid that is bound to a synthetic nanocarrier.

  The nucleic acid can be DNA or RNA such as mRNA. In embodiments, the compositions of the invention include complement, such as the full-length complement of any of the nucleic acids provided herein, or altered tissue (due to altered genetic code). In embodiments, the nucleic acid is an expression vector that can be transcribed when transfected into a cell line. In embodiments, the expression vector may include, in particular, a plasmid, retrovirus, or adenovirus. Nucleic acids are isolated or synthesized using standard molecular biology techniques, for example, using the polymerase chain reaction to produce a nucleic acid fragment that is then purified and cloned into an expression vector. Can be done. Additional techniques useful for the practice of the present invention are described in John Wiley and Sons, Inc. Current Protocols in Molecular Biology 2007 by; Molecular Cloning: A Laboratory Manual (Third Edition) Joseph Sambrook, Peter MacCallum Cancer Institute, Melbourne, Australia; David Russell, University of Texas Southwestern Medical Center, Dallas, can be found in Cold Spring Harbor .

  In embodiments, the immunosuppressive agents provided herein are bound to a synthetic nanocarrier. In a preferred embodiment, the immunosuppressive agent is an element added to the materials that make up the structure of the synthetic nanocarrier. For example, in one embodiment, when the synthetic nanocarrier is composed of one or more polymers, the immunosuppressive agent is a compound that is added to and bound to one or more polymers. As another example, in one embodiment, when the synthetic nanocarrier is composed of one or more lipids, an immunosuppressive agent is similarly added and bound to the one or more lipids. In embodiments, such as where the synthetic nanocarrier material also provides an immunosuppressive (eg, tolerogenic) effect, the immunosuppressive agent is in addition to the synthetic nanocarrier material that provides the immunosuppressive (eg, tolerogenic) effect. It is an existing element.

  Other exemplary immunosuppressive agents include, but are not limited to, small molecule drugs, natural products, antibodies (eg, antibodies to CD20, CD3, CD4), biologic based drugs, carbohydrate based drugs, nano Particles, liposomes, RNAi, antisense nucleic acids, aptamers, methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3; tacrolimus (FK506) and the like. Additional immunosuppressive agents are known to those skilled in the art and the invention is not limited in this regard.

  “Inflammatory disease” means any disease, disorder or condition in which undesirable inflammation occurs.

  An “load” of immunosuppressant or antigen is the amount of immunosuppressant or antigen bound to the synthetic nanocarrier, based on the total weight (weight / weight) of the material in the total synthetic nanocarrier. In general, the load is calculated as the average of the entire population of synthetic nanocarriers. In one embodiment, the immunosuppressant loading is 0.0001% to 50%, averaged over the entire first population of synthetic nanocarriers. In another embodiment, the antigen loading is 0.0001% to 50% on average across the first and / or second population of synthetic nanocarriers. In yet another embodiment, the immunosuppressant and / or antigen load is between 0.01% and 20%. In a further embodiment, the immunosuppressant and / or antigen load is between 0.1% and 10%. In still other embodiments, the immunosuppressant and / or antigen load is between 1% and 10%. In yet another embodiment, the immunosuppressive agent and / or antigen loading is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.1%, averaged over the entire population of synthetic nanocarriers. 4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17% , At least 18%, at least 19% or at least 20%. In still other embodiments, the immunosuppressive agent and / or antigen loading is 0.1%, 0.2%, 0.3%, 0.4%, 0%, averaged over the entire population of synthetic nanocarriers. .5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%. In certain embodiments of the above embodiments, the immunosuppressant and / or antigen load is 25% or less on average over the entire population of synthetic nanocarriers. In an embodiment, the load is calculated as described in the examples.

  In any embodiment of the provided compositions and methods, the load is calculated as follows: about 3 mg of synthetic nanocarrier is collected and centrifuged to separate the supernatant from the synthetic nanocarrier pellet. Acetonitrile is added to the pellet and the sample is sonicated and centrifuged to remove any insoluble material. The supernatant and pellet are injected into RP-HPLC and the absorbance is read at 278 nm. The μg found in the pellet is used to calculate% uptake (load), and the μg in the supernatant and pellet is used to calculate the total μg recovered.

  A “maintenance dose” is administered to a subject after the initial dose has resulted in an immunosuppressive (eg, tolerogenic) response in the subject to sustain the desired immunosuppressive (eg, tolerogenic) response. Refers to the dose to be administered. A maintenance dose, for example, maintains a tolerogenic effect obtained after an initial dose, prevents an undesirable immune response in a subject, or an undesirable immune response in which the subject contains an undesirable level of immune response Can be a dose that prevents becoming a subject at risk of developing. In certain embodiments, the maintenance dose is a dose sufficient to sustain an appropriate level of the desired immune response.

  “Maximum dimension of a synthetic nanocarrier” means the maximum dimension of the nanocarrier measured along any axis of the synthetic nanocarrier. “Minimum dimension of a synthetic nanocarrier” means the smallest dimension of a synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, in a spherical synthetic nanocarrier, the maximum and minimum dimensions of the synthetic nanocarrier will be substantially the same and the size of its diameter. Similarly, for cubic synthetic nanocarriers, the smallest dimension of a synthetic nanocarrier is the smallest of its height, width or length, while the largest dimension of a synthetic nanocarrier is its height, width or length. It will be the largest of them. In one embodiment, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample is 100 nm or more, based on the total number of synthetic nanocarriers in the sample. . In one embodiment, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample, based on the total number of synthetic nanocarriers in the sample, is 5 μm or less. . Preferably, based on the total number of synthetic nanocarriers in the sample, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample is greater than 110 nm, more preferably More than 120 nm, more preferably more than 130 nm, even more preferably more than 150 nm. The aspect ratio of the maximum and minimum dimensions of the synthetic nanocarrier of the present invention can vary depending on the embodiment. For example, the aspect ratio of the largest dimension to the smallest dimension of the synthetic nanocarrier is from 1: 1 to 1,000,000: 1, preferably from 1: 1 to 100,000: 1, more preferably from 1: 1 to 10,000. : 1, more preferably 1: 1 to 1000: 1, even more preferably 1: 1 to 100: 1, even more preferably 1: 1 to 10: 1. Preferably, based on the total number of synthetic nanocarriers in the sample, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample is 3 μm or less, more preferably It is 2 μm or less, more preferably 1 μm or less, more preferably 800 nm or less, more preferably 600 nm or less, and even more preferably 500 nm or less. In a preferred embodiment, based on the total number of synthetic nanocarriers in the sample, a minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in the sample is 100 nm or more, more Preferably it is 120 nm or more, More preferably, it is 130 nm or more, More preferably, it is 140 nm or more, More preferably, it is 150 nm or more. Measurement of synthetic nanocarrier dimensions (eg, diameter) suspends synthetic nanocarriers in a liquid (usually aqueous) medium and uses dynamic light scattering (DLS) (eg, using a Brookhaven ZetaPALS instrument). Can be obtained. For example, a suspension of synthetic nanocarrier is diluted from aqueous buffer into purified water to obtain a final synthetic nanocarrier suspension concentration of about 0.01-0.1 mg / mL. The diluted suspension may be prepared directly in or transferred to a suitable cuvette for DLS analysis. The cuvette is then placed in DLS and allowed to equilibrate to a controlled temperature and then scanned for a sufficient amount of time to be stable and reproducible based on appropriate inputs for media viscosity and sample refractive index. Get a good distribution. Next, the effective diameter, or average of the distribution, is reported. The “dimension” or “size” or “diameter” of a synthetic nanocarrier refers to the average particle size distribution obtained using dynamic light scattering.

  “MHC” refers to the major histocompatibility complex, large genomic region or gene family found in most vertebrates that encode MHC molecules that represent fragments or epitopes of processed proteins on the cell surface. Presentation of MHC: peptides on the cell surface allows monitoring by immune cells, usually T cells. There are two general classes of MHC molecules, class I and class II. In general, class I MHC molecules are found in nucleated cells and present peptides to cytotoxic T cells. Class II MHC molecules are found in certain immune cells, mainly macrophages, B cells and dendritic cells, collectively known as professional APCs. The best known genes in the MHC region are the subsets that encode antigen presenting proteins on the cell surface. In humans, these genes are called human leukocyte antigen (HLA) genes.

  “Non-methoxy terminated polymer” means a polymer having at least one terminus terminated with a moiety other than methoxy. In certain embodiments, the polymer has at least two ends that terminate with moieties other than methoxy. In other embodiments, the polymer does not have a methoxy terminated end. “Non-methoxy terminated pluronic polymer” means a polymer other than a linear pluronic polymer having methoxy at both ends. The polymer nanoparticles provided herein can comprise a non-methoxy terminated polymer or a non-methoxy terminated pluronic polymer.

  “Pharmaceutically acceptable excipient” means a pharmacologically inert material used in conjunction with the described synthetic nanocarriers to formulate the compositions of the invention. Pharmaceutically acceptable excipients include saccharides (glucose, lactose, etc.), preservatives such as antibacterial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers. Various materials known in the art are included, including but not limited to.

  “Protocol” refers to any regime of administration of one or more substances to a subject. The dosage regimen may include the amount, frequency and / or form of administration. In certain embodiments, such protocols can be used to administer one or more compositions of the present invention to one or more subjects. The immune response in these subjects can then be evaluated to determine whether the protocol was effective in reducing the unwanted immune response or generating the desired immune response (eg, promoting a tolerogenic effect). . Any other therapeutic and / or prophylactic effect can also be evaluated instead of or in addition to the immune response described above. Whether a protocol had the desired effect can be determined using any of the methods provided herein or known in the art. To determine whether a particular immune cell, cytokine, antibody, etc. has been reduced, generated, activated, etc., for example, a population of cells is administered a composition provided herein according to a particular protocol. Obtained from a subject. Useful methods for detecting the presence and / or number of immune cells include, but are not limited to, flow cytometry (eg, FACS) and immunohistochemistry. Antibodies and other binding agents are commercially available for specific staining of immune cell markers. Such kits typically include staining reagents for multiple antigens that allow FACS-based detection, separation and / or quantification of the desired cell population from a heterogeneous cell population.

  “Providing a subject” causes a clinician to contact the subject and administer the composition provided herein to the subject or to perform the method provided herein on the subject. Any action or series of actions. Preferably, the subject is a subject in need of the tolerogenic immune response provided herein. The action or sequence of actions can be performed directly on its own, or indirectly, but not exclusively, by an unrelated third party who acts by trusting in human behavior.

  “Subject” refers to warm-blooded mammals such as humans and primates; birds; domestic animals or livestock such as cats, dogs, sheep, goats, cows, horses and pigs; experiments such as mice, rats and guinea pigs Animals; fish; means reptiles; animals including zoo animals and wild animals.

  “Synthetic nanocarrier” means an individual object that is not found in nature and has at least one dimension that is 5 μm or less in size. Albumin nanoparticles are generally included as synthetic nanocarriers, but in certain embodiments, synthetic nanocarriers do not include albumin nanoparticles. In embodiments, the synthetic nanocarrier of the present invention does not include chitosan. In other embodiments, the synthetic nanocarriers of the invention are not lipid-based nanoparticles. In further embodiments, the synthetic nanocarriers of the present invention do not include phospholipids.

  Synthetic nanocarriers include, but are not limited to, one or more lipid-based nanoparticles (also referred to herein as lipid nanoparticles, ie, nanoparticles in which the majority of the material making up the structure is lipid) , Polymer nanoparticles, metal nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles (ie composed primarily of viral structural proteins but are not infectious or have low infectivity) Particles), peptide or protein-based particles (also referred to herein as protein particles, ie particles in which the majority of the material comprising the structure is peptides or proteins) (such as albumin nanoparticles) and / or lipid-polymer nano Nanoparticles formed using a combination of nanomaterials such as particles That. Synthetic nanocarriers can be in a variety of different shapes including, but not limited to, spherical, cubic, conical, oblong, cylindrical, donut shaped, and the like. The synthetic nanocarrier according to the present invention comprises one or more surfaces. Exemplary synthetic nanocarriers that can be adapted for use in the practice of the present invention are: (1) Gref et al. Biodegradable nanoparticles disclosed in US Pat. No. 5,543,158 to (2) Saltzman et al. U.S. Patent Application Publication No. 20060002852 to polymer nanoparticles, (3) DeSimon et al. US Patent Application Publication No. 20090028910 to lithographically composed nanoparticles, (4) von Andrian et al. Disclosed in International Publication No. 2009/051837 pamphlet, (5) Penades et al. Nanoparticles disclosed in US Patent Application Publication No. 2008/0145441 to (6) de los Rios et al. Protein nanoparticles disclosed in US Patent Application Publication No. 20090226525 to (7) Sebel et al. Virus-like particles disclosed in US Patent Application Publication No. 20060226552 to (8) Bachmann et al. (9) a virus-like particle disclosed in International Publication No. 20110047839A1 or International Publication No. 2009106999A2, (10) P.I. Paulicelli et al. "Surface-modified PLGA-based Nanoparticulates that can effectively associate and deliver virus-like particles" Nanomedicine. 5 (6): 843-853 (2010), nanoprecipitated nanoparticles, or (11) apoptotic cells, apoptotic bodies disclosed in US Patent Application Publication No. 2002/0086049 Or synthetic or semi-synthetic analogues. In embodiments, the synthetic nanocarrier has a 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7 aspect ratio, or a 1:10 aspect ratio. May have a ratio.

  Synthetic nanocarriers according to the present invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, do not include surfaces with hydroxyl groups that activate complement, or alternatively are not hydroxyl groups that activate complement Includes a surface consisting essentially of parts. In a preferred embodiment, the synthetic nanocarrier according to the present invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, does not include a surface that substantially activates complement or instead substantially comprises complement. Including a surface consisting essentially of non-activated portions. In a more preferred embodiment, the synthetic nanocarrier according to the present invention having a minimum dimension of about 100 nm or less, preferably 100 nm or less, does not include a surface that activates complement, or alternatively from a portion that does not activate complement. Includes a surface that essentially consists of. In embodiments, the synthetic nanocarrier excludes virus-like particles. In embodiments, the synthetic nanocarrier has a 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 5, 1: 7 aspect ratio, or a 1:10 aspect ratio. May have a ratio.

  “T cell antigen” means CD4 + T cell antigen, CD8 + cell antigen or CD1d restricted antigen. A “CD4 + T cell antigen” is recognized by an immune response in CD4 + T cells, eg, by presentation of any antigen bound by, or a portion of, a class II major histocompatibility complex molecule (MHC). By antigen specifically recognized by the T cell receptor in the cell. A “CD8 + T cell antigen” is recognized by the immune response in CD8 + T cells, eg, by presentation of any antigen or portion thereof bound to a class I major histocompatibility complex molecule (MHC), resulting in CD8 + T cells. By antigen specifically recognized by the T cell receptor in the cell. By “CD1d-restricted antigen” is meant an antigen that binds to, is complexed with, or comprises one or more epitopes presented by a CD1d molecule. In general, CD1d-restricted T cell antigens are lipids presented to invariant NKT cells. CD1d-restricted T cell antigens include α-galactosylceramide (α-GalCer), α-linked glycosphingolipid (derived from Sphingomonas spp.), Galactosyl diacylglycerol (Borrelia burgdorferi) ), Lipophosphoglycan (derived from Leishmania donovani), endogenous or exogenous β-glucosylceramide, and phosphatidylinositol tetramannoside (PIM4) (Mycobacterium leprae) One or more lipids, or glycolipids, including but not limited to. For additional lipids and / or glycolipids useful as CD1d restricted antigens, see V. Cerundolo et al. , “Harnessing invious NKT cells in vacuation strategies.” Nature Rev Immun, 9: 28-38 (2009). In certain embodiments, an antigen that is a T cell antigen is also a B cell antigen. In other embodiments, the T cell antigen is also not a B cell antigen. T cell antigens are generally proteins or peptides, but may be other molecules such as lipids and glycolipids.

  “Therapeutic protein” refers to any protein or protein-based therapy that can be administered to a subject and have a therapeutic effect. Such therapies include protein replacement therapy and protein addition therapy. Such therapies also include administration of exogenous or heterologous proteins, antibody therapy, and cell therapy or cell-based therapy. Therapeutic proteins include enzymes, enzyme cofactors, hormones, blood clotting factors, cytokines, growth factors, monoclonal antibodies and polyclonal antibodies. Examples of other therapeutic proteins are provided elsewhere herein. The therapeutic protein may be produced in, on or by the cell, obtained from or administered in the form of such a cell. In embodiments, the therapeutic protein is produced in or on a mammalian cell, insect cell, yeast cell, bacterial cell, plant cell, transgenic animal cell, transgenic plant cell, and the like. The therapeutic protein can be produced by recombinant techniques in such cells. The therapeutic protein can be produced in or on virally transformed cells. The therapeutic protein can also be produced in, on or by autologous cells that have been transfected, transduced or otherwise engineered to express it. Alternatively, the therapeutic protein can be administered as a nucleic acid or by introducing the nucleic acid into a virus, VLP, liposome, or the like. Alternatively, the therapeutic protein can be obtained from such a form and administered as the therapeutic protein itself. Thus, a subject includes any subject that has been administered, is being administered, or is scheduled to be administered any of the above. Such a subject may be a gene therapy; an autologous cell that has been transfected, transduced or otherwise manipulated to express a therapeutic protein, polypeptide or peptide; or a therapeutic protein, polypeptide Or includes a subject that has been, is or will be administered cells expressing the peptide.

  “Therapeutic protein antigen” means an antigen associated with a therapeutic protein, such antigen or a portion thereof may be presented for recognition by cells of the immune system, such as an undesirable immune response against the therapeutic protein (eg, , Promotion of antibodies specific for therapeutic proteins). Therapeutic protein antigens generally include proteins, polypeptides, peptides, lipoproteins, or are contained or expressed in, on or by a cell.

  By “tolerogenic immune response” is meant any immune response that can result in specific immunosuppression for an antigen or cells, tissues, organs, etc. that express such antigen. Such an immune response includes any reduction, delay or inhibition of an unwanted immune response specific for an antigen or a cell, tissue, organ, etc. that expresses such an antigen. Such an immune response also includes any stimulation, generation, induction, promotion or mobilization of the desired immune response specific for the antigen or cells, tissues, organs, etc. that express such antigen. Thus, a tolerogenic immune response includes the absence or reduction of an unwanted immune response to an antigen that can be mediated by antigen-reactive cells as well as the presence or promotion of suppressor cells. Tolerogenic immune responses provided herein include immune tolerance. “Generate a tolerogenic immune response” refers to the generation of any of the above immune responses specific for an antigen or a cell, tissue, organ, etc. that expresses such an antigen. The tolerogenic immune response can be the result of MHC class I restricted presentation and / or MHC class II restricted presentation and / or B cell presentation and / or presentation by CD1d.

  Tolerogenic immune responses include any reduction, delay or inhibition of CD4 + T cell, CD8 + T cell or B cell proliferation and / or activity. A tolerogenic immune response also includes a decrease in antigen-specific antibody production. A tolerogenic immune response can include any response that results in stimulation, induction, production or mobilization of regulatory cells such as CD4 + Treg cells, CD8 + Treg cells, Breg cells. In certain embodiments, the tolerogenic immune response is one that results in a conversion to a regulatory phenotype characterized by the production, induction, stimulation or mobilization of regulatory cells.

  A tolerogenic immune response also includes any response that results in stimulation, production or mobilization of CD4 + Treg cells and / or CD8 + Treg cells. CD4 + Treg cells express the transcription factor FoxP3 and can inhibit inflammatory responses and autoimmune inflammatory diseases (Human regulatory T cells in autoimmune diseases. Cvetanovich GL, Hafler DA. Curr Opin Immun. 20; 753-60.Regularity T cells and autoimmunity.Vila J, Isaacs JD, Anderson AE. Curr Opin Hematol.2009 Jul; 16 (4): 274-9). Such cells also inhibit T cells from helping B cells and induce tolerance to both self and foreign antigens (Therapeutic approaches to allergy and autoimmunity based on Foxp3 + regulatory T-cell activation.) M, Wing K, Sakaguchi S. J Allergy Clin Immunol. 2009 Apr; 123 (4): 749-55). CD4 + Treg cells recognize antigens when presented by class II proteins in APC. CD8 + Treg cells that recognize antigens presented by class I (and Qa-1) also inhibit T cells from helping B cells and induce antigen tolerance to both self and foreign antigens Can lead to the activation of. Disruption of Qa-1 interaction with CD8 + Treg cells has been shown to dysregulate the immune response, resulting in the formation of autoantibodies and the development of systemic lupus erythematosus that destroys autoimmunity (Kim et al. , Nature.2010 Sep 16,467 (7313): 328-32). CD8 + Treg cells have also been shown to inhibit models of autoimmune inflammatory diseases including rheumatoid arthritis and colitis (CD4 + CD25 + regulatory T cells in autoimmune arthritis. Oh S, Rankin AL, Caton AJ. ImmunoReJ. Immunol 20). 233 (1): 97-111.Regularity T cells in inflammatory bow disease.Boden EK, Snapper SB.Curr Opin Gastroenterol.2008 Nov; 24 (6): 733-41). In certain embodiments, provided compositions can effectively provide both types of responses (CD4 + Treg and CD8 + Treg). In other embodiments, FoxP3 can be induced in other immune cells such as macrophages, iNKT cells, and the compositions provided herein can result in one or more of these responses as well.

  Tolerogenic immune responses include, but are not limited to, induction of regulatory cytokines such as Treg cytokines; induction of inhibitory cytokines; inflammatory cytokines (eg, IL-4, IL-1b, IL-5, TNF) -Α, IL-6, GM-CSF, IFN-γ, IL-2, IL-9, IL-12, IL-17, IL-18, IL-21, IL-22, IL-23, M-CSF , Inhibition of C-reactive protein, acute phase protein, chemokine (eg MCP-1, RANTES, MIP-1α, MIP-1β, MIG, ITAC or IP-10), anti-inflammatory cytokines (eg IL-4, IL-13, IL-10, etc.), chemokines (eg CCL-2, CXCL8), proteases (eg MMP-3, MMP-9), leukotri (Eg, CysLT-1, CysLT-2), prostaglandin (eg, PGE2) or histamine; inhibition of Th17, Th1 or Th2 immune response; effector cell specific cytokine: Th17 ( For example, inhibition of IL-17, IL-25), Th1 (IFN-γ), Th2 (eg, IL-4, IL-13); inhibition of Th1-, Th2- or TH17-specific transcription factors; effector T Inhibition of cell proliferation; inhibition of apoptosis of effector T cells; induction of tolerogenic dendritic cell-specific genes, induction of FoxP3 expression, inhibition of IgE-induced or IgE-mediated immune responses; antibody responses (eg antigen-specific Antibody production); inhibition of T helper cell response; TGF-β and / or Production of IL-10; inhibition of autoantibody effector function (e.g., depletion of cells, inhibition of damage or complement activation of a cell or tissue), and also like.

  Any of the above may be measured in vivo or in vitro in one or more animal models. Such in vivo or in vitro measurements are well known to those skilled in the art. Undesirable or tolerogenic immune responses are e.g. methods for assessing the number and / or function of immune cells, tetramer analysis, ELISPOT, cytokine expression, cytokine secretion, cytokine expression profiling, gene expression profiling, protein expression It can be monitored using flow cytometry analysis of profiling, analysis of cell surface markers, PCR based detection using immune cell receptor genes, etc. (T. Clay et al., “Assays for Monitoring Cellular Immunity Response”. to Active Immunotherapy of Cancer ”Clinical Cancer Research 7: 1127-1135 (2001). Thailand). Undesirable or tolerogenic immune responses can also be monitored using, for example, methods of assessing protein levels in plasma or serum, immune cell proliferation and / or functional assays, and the like. In certain embodiments, the tolerogenic immune response can be monitored by assessing the induction of FoxP3. Furthermore, specific methods are described in more detail in the examples.

  Preferably, the tolerogenic immune response results in the onset, progression or pathology inhibition of the diseases, disorders or conditions described herein. It is determined using an animal model of such a disease, disorder or condition whether the composition of the invention can result in the onset, progression or inhibition of the disease, disorder or condition described herein. Can be done.

  In certain embodiments, the reduction of an unwanted immune response or the generation of a tolerogenic immune response is assessed by measuring clinical endpoints, clinical efficacy, clinical symptoms, disease biomarkers and / or clinical scores. obtain. An undesirable immune response or tolerogenic immune response can also be assessed using a diagnostic test to assess the presence or absence of a disease, disorder or condition provided herein. Undesirable immune responses can be further assessed by methods of measuring therapeutic protein levels and / or function in a subject. In an embodiment, a method for monitoring or assessing an undesired allergic response comprises assessing an allergic response in a subject due to skin reaction and / or allergen specific antibody production.

  In certain embodiments, monitoring or evaluation of the generation of an undesirable or tolerogenic immune response in a subject is performed prior to administration of the composition of synthetic nanocarriers provided herein and / or implantable transplants. It may be prior to administration of the strip or therapeutic protein or exposure to the allergen. In other embodiments, the assessment of the generation of an undesirable or tolerogenic immune response is performed after administration of the composition of the synthetic nanocarrier provided herein and / or for an implantable graft or treatment It may be after administration of the protein or exposure to the allergen. In certain embodiments, the evaluation is performed after administration of the composition of the synthetic nanocarrier, but prior to administration of the implantable graft or therapeutic protein or exposure to the allergen. In other embodiments, the assessment is performed after administration of the implantable graft or therapeutic protein or exposure to the allergen, but prior to administration of the composition. In still other embodiments, the assessment is performed prior to both administration of the synthetic nanocarrier and administration of the implantable graft or therapeutic protein or exposure to the allergen, while in still other embodiments, the assessment is performed. , Both after administration of the synthetic nanocarrier and administration of the implantable graft or therapeutic protein or exposure to the allergen. In further embodiments, the assessment is performed both before and after administration of the synthetic nanocarrier and / or administration of the implantable graft or therapeutic protein or exposure to the allergen. In yet other embodiments, a subject such as a subject whose desirable immune condition is or is at risk of suffering from an inflammatory disease, autoimmune disease, allergy, organ or tissue rejection or graft-versus-host disease In order to determine that it is maintained in, the assessment is performed more than once per subject. Whether the other subject has or has been administered a therapeutic protein that has undergone or will undergo a transplant, as well as a therapeutic protein that has, has, or is expected to have an undesirable immune response Or a subject that is to be administered.

  The antibody response can be assessed by measuring one or more antibody titers. “Antibody titer” means a measurable level of antibody production. Methods for measuring antibody titer are known in the art and include enzyme linked immunosorbent assay (ELISA). In embodiments, the antibody response can be quantified, for example, as the number of antibodies, antibody concentration or titer. The value may be an absolute value or a relative value. Assays for quantifying antibody responses include antibody capture assays, enzyme-linked immunosorbent assay (ELISA), inhibitory phase adsorption assay (ILPAA), rocket immunoelectrophoresis (RIE) assay and linear immunoelectrophoresis (LIE). An assay. When an antibody response is compared to another antibody response, the same type of quantitative value (eg, titer) and measurement method (eg, ELISA) are preferably used to make the comparison.

  An ELISA method for measuring antibody titer, such as a typical sandwich ELISA, comprises the following steps (i) ELISA plate coating material so that the antibody target of interest is bound to a substrate polymer or other suitable material (Ii) preparing a coating material in an aqueous solution (such as PBS) and delivering the coating material solution to the wells of the multi-well plate to deposit the coating on the multi-well plate overnight; (Iii) thoroughly washing the multiwell plate with a wash buffer (such as 0.05% Tween-20 in PBS) to remove excess coating material, (iv) a diluent solution (10% in PBS To block the plate for non-specific binding by applying fetal bovine serum, etc.) , (V) washing the blocking / diluent solution from the plate with wash buffer, (vi) diluting serum sample containing antibody and appropriate standard (positive control) with diluent as needed Obtaining a concentration that suitably satisfies an ELISA response, (vii) serially diluting a plasma sample in a multiwell plate to cover a range of concentrations suitable for generating an ELISA response curve, (viii) ) Incubating the plate to obtain antibody-target binding; (ix) washing the plate with a wash buffer to remove antibody not bound to the antigen; (x) binding the primary antibody. Adding a secondary detection antibody, such as a possible biotin-conjugated detection antibody, at the appropriate concentration in the same diluent, (xi) plate with the applied detection antibody (Xiii) a step of adding an enzyme such as streptavidin-HRP (horseradish peroxidase) capable of binding to biotin, which is found in a biotinylated antibody, and incubating, (xiii) Washing the well plate, (xiv) adding a substrate (such as TMB solution) to the plate, (xv) applying a stop solution (such as 2N sulfuric acid) when color development is complete, (xvi) a specific wavelength of the substrate Reading the optical density of the plate wells (subtracting the reading at 570 nm from the reading at 450 nm), (xvi) applying a suitable multiparameter curve fit to the data, and calculating the maximum OD for the plate standard Define half-effect concentration (EC50) as the concentration in the curve where half-value is obtained It can consist of the process to do.

  “Implantable graft” refers to biomaterials, such as cells, tissues and organs (whole organs or partial organs) that can be administered to a subject. Implantable grafts can be obtained from biomaterials such as organs, tissues, skin, bones, nerves, tendons, neurons, blood vessels, fats, corneas, pluripotent cells, differentiated cells (in vivo or in vitro). Or autologous graft, xenograft, or xenograft. In certain embodiments, the implantable implant is formed from, for example, cartilage, bone, extracellular matrix, or collagen matrix. An implantable implant may also be a single cell, a cell and a suspension of cells in tissues and organs that can be transplanted. Transplantable cells usually have a therapeutic function, eg, a function that is absent or reduced in the recipient subject. Some non-limiting examples of transplantable cells are beta cells, hepatocytes, hematopoietic stem cells, neural stem cells, neurons, glial cells, or myelinating cells. Transplantable cells can be unmodified cells, eg, cells obtained from a donor subject and usable for transplantation without any genetic modification or epigenetic modification. In other embodiments, the transplantable cell is a modified cell, e.g., a cell obtained from a subject with a genetic defect that has been corrected for a genetic defect, or a cell derived from a reprogrammed cell, e.g., It can be a differentiated cell derived from a cell obtained from a subject.

  “Transplant” refers to transplanting an implantable graft (eg, from a donor subject, from an in vitro source (eg, a differentiated autologous cell or heterologous somatic cell or derived pluripotent cell)). Refers to the process of moving (moving) to and / or from one body part of the same subject to another body part.

  An “unwanted immune response” results from exposure to an antigen and promotes or exacerbates a disease, disorder or condition (or symptom thereof) provided herein, or a disease, disorder provided herein. Or refers to any undesirable immune response that shows signs of pathology. Such an immune response generally has an adverse effect on the health of the subject or is indicative of an adverse effect on the health of the subject.

C. Compositions of the Invention Provided herein are compositions of synthetic nanocarriers and immunosuppressive agents that can provide an immunosuppressive effect when administered to a subject. Preferably, the composition also includes an antigen and the immunosuppressive effect is specific to the antigen and thus tolerogenic effects. Such compositions are any of the tolerogenic responses provided herein, such as stimulation, induction, production or mobilization of regulatory cells (eg, CD4 ⁺ Treg cells and / or CD8 ⁺ Treg cells). Can bring In certain embodiments, the tolerogenic immune response is one that results in a conversion to a regulatory phenotype characterized by production, induction, stimulation or mobilization of regulatory cells (eg, Treg cells).

  As described above, synthetic nanocarriers are designed to include immunosuppressive agents, in certain embodiments, antigens for which a tolerogenic effect is desired. A variety of synthetic nanocarriers can be used in accordance with the present invention. In certain embodiments, the synthetic nanocarrier is a sphere or spheroid. In certain embodiments, the synthetic nanocarrier is flat or plate-like. In certain embodiments, the synthetic nanocarrier is cubic or cubic. In certain embodiments, the synthetic nanocarrier is elliptical or oval. In certain embodiments, the synthetic nanocarrier is cylindrical, conical, or pyramidal.

  In certain embodiments, it may be desirable to use a population of synthetic nanocarriers that is relatively uniform in size, shape, and / or composition so that each synthetic nanocarrier has similar properties. For example, based on the total number of synthetic nanocarriers, at least 80%, at least 90%, or at least 95% of the synthetic nanocarriers are 5%, 10%, or 20% of the average diameter or average dimension of the synthetic nanocarriers. It may have a minimum or maximum dimension within the range. In certain embodiments, the population of synthetic nanocarriers may be heterogeneous with respect to size, shape, and / or composition.

  Synthetic nanocarriers can be solid or hollow and can include one or more layers. In some embodiments, each layer has a unique composition and unique properties compared to other layers. By way of example only, a synthetic nanocarrier may have a core / shell structure, where the core is one layer (eg, a polymer core) and the shell is a second layer (eg, a lipid core). Double layer or single layer). Synthetic nanocarriers can include multiple different layers.

  In certain embodiments, the synthetic nanocarrier can optionally include one or more lipids. In certain embodiments, the synthetic nanocarrier can comprise a liposome. In certain embodiments, the synthetic nanocarrier can comprise a lipid bilayer. In certain embodiments, the synthetic nanocarrier can comprise a lipid monolayer. In certain embodiments, the synthetic nanocarrier can comprise micelles. In certain embodiments, a synthetic nanocarrier can include a core comprising a polymer matrix surrounded by a lipid layer (eg, lipid bilayer, lipid monolayer, etc.). In certain embodiments, the synthetic nanocarrier is a non-polymeric core (eg, metal particles, quantum dots, ceramic particles, bone particles, virus particles, surrounded by a lipid layer (eg, lipid bilayer, lipid monolayer, etc.) Protein, nucleic acid, carbohydrate, etc.).

  In certain embodiments, the synthetic nanocarrier does not include a polymer component. In certain embodiments, synthetic nanocarriers can include metal particles, quantum dots, ceramic particles, and the like. In certain embodiments, the non-polymeric synthetic nanocarrier is a collection of non-polymeric components, such as a collection of metal atoms (eg, gold atoms).

  In certain embodiments, the synthetic nanocarrier can optionally include one or more amphiphiles. In certain embodiments, the amphiphile can facilitate the production of synthetic nanocarriers having increased stability, improved uniformity, or increased viscosity. In certain embodiments, the amphiphile can be bound to the inner surface of a lipid membrane (eg, lipid bilayer, lipid monolayer, etc.). Many amphiphiles known in the art are suitable for use in making synthetic nanocarriers according to the present invention. Such amphiphiles include, but are not limited to, phosphoglycerides; phosphatidylcholines; dipalmitoylphosphatidylcholine (DPPC); dioleoylphosphatidylethanolamine (DOPE); dioleoyloxypropyltriethylammonium (DOTMA); Phosphatidylcholine; cholesterol; cholesterol ester; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; palmitic acid or olein Surface active fatty acids such as acids; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; sorbi N-trioleate (Span® 85) glycocholate; sorbitan monolaurate (Span® 20); polysorbate 20 (Tween® 20); polysorbate 60 (Tween® 60); polysorbate 65 (Tween® 65); polysorbate 80 (Tween® 80); polysorbate 85 (Tween® 85); polyoxyethylene monostearate; surfactin; poloxamer; sorbitan such as sorbitan trioleate Fatty acid ester; lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid; Dicetyl phosphate; dipalmitoyl phosphatidylglycerol; stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecyl stearate; isopropyl myristate; tyloxapol; poly (ethylene glycol) 5000-phosphatidylethanolamine; Poly (ethylene glycol) 400-monostearate; phospholipids; synthetic and / or natural detergents with high surfactant properties; deoxycholates; cyclodextrins; chaotropic salts; ion-pairing agents; The amphiphile component may be a mixture of different amphiphiles. One skilled in the art will recognize that this is an exemplary rather than a comprehensive list of materials having surfactant activity. Any amphiphile can be used to produce the synthetic nanocarrier used in accordance with the present invention.

  In certain embodiments, the synthetic nanocarrier can optionally include one or more carbohydrates. Carbohydrates can be natural or synthetic. The carbohydrate may be a derivatized natural carbohydrate. In certain embodiments, the carbohydrate comprises glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellobiose, mannose, xylose, arabinose, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid. Including monosaccharide or disaccharide including but not limited to. In certain embodiments, the carbohydrate is pullulan, cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, hydroxyethyl starch, carrageenan, glycone (Glycon), amylose, chitosan, N, O-carboxymethylchitosan, algin and alginic acid, starch, chitin, inulin, konjac, glucomannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan It is not limited to polysaccharides. In embodiments, the synthetic nanocarriers of the invention do not include (or specifically exclude) carbohydrates such as polysaccharides. In certain embodiments, the carbohydrate may include carbohydrate derivatives such as sugar alcohols including but not limited to mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.

  In certain embodiments, synthetic nanocarriers can include one or more polymers. In certain embodiments, the synthetic nanocarrier comprises one or more polymers that are non-methoxy terminated pluronic polymers. In certain embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of the polymer comprising the synthetic nanocarrier, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight / weight) is non-methoxy terminal pluronic It is a polymer. In certain embodiments, all of the polymers making up the synthetic nanocarrier are non-methoxy terminated pluronic polymers. In certain embodiments, the synthetic nanocarrier comprises one or more polymers that are non-methoxy terminated polymers. In certain embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of the polymer comprising the synthetic nanocarrier, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight / weight) is a non-methoxy terminated polymer It is. In certain embodiments, all of the polymers comprising the synthetic nanocarrier are non-methoxy terminated polymers. In certain embodiments, the synthetic nanocarrier comprises one or more polymers that do not comprise a pluronic polymer. In certain embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of the polymer comprising the synthetic nanocarrier, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight / weight) contain pluronic polymer Absent. In certain embodiments, all of the polymers comprising the synthetic nanocarrier do not include a pluronic polymer. In certain embodiments, such polymers can be surrounded by a coating layer (eg, liposomes, lipid monolayers, micelles, etc.). In certain embodiments, various elements of the synthetic nanocarrier can be combined with a polymer.

  Immunosuppressive agents and / or antigens can be bound to synthetic nanocarriers by any of several methods. In general, binding can be the result of binding of an immunosuppressant and / or antigen to a synthetic nanocarrier. This binding allows immunosuppressive agents and / or antigens to be bound to and / or included (encapsulated) in the surface of the synthetic nanocarrier. However, in certain embodiments, the immunosuppressive agent and / or antigen is encapsulated by the synthetic nanocarrier as a result of the structure of the synthetic nanocarrier rather than binding to the synthetic nanocarrier. In preferred embodiments, the synthetic nanocarrier comprises a polymer provided herein, and an immunosuppressive agent and / or antigen is attached to the polymer.

  When binding occurs as a result of binding of an immunosuppressive agent and / or antigen to a synthetic nanocarrier, binding can occur via a binding moiety. The binding moiety can be any moiety where an immunosuppressant and / or antigen is bound to a synthetic nanocarrier. Such moieties include covalent bonds, such as amide bonds or ester bonds, and separate molecules that bind (covalently or non-covalently) immunosuppressive agents and / or antigens to synthetic nanocarriers. Such molecules include linkers or polymers or units thereof. For example, the binding moiety can include a charged polymer to which the immunosuppressant and / or antigen is electrostatically bound. As another example, a binding moiety can include a polymer or unit thereof to which it is covalently bonded.

  In preferred embodiments, the synthetic nanocarrier comprises a polymer provided herein. These synthetic nanocarriers can be completely polymers or can be a mixture of polymers and other materials.

  In certain embodiments, the polymers of the synthetic nanocarrier are combined to form a polymer matrix. In some of these embodiments, components such as immunosuppressive agents or antigens can be covalently bound to one or more polymers of the polymer matrix. In certain embodiments, the covalent bond can be made through a linker. In certain embodiments, the component can be non-covalently bound to one or more polymers of the polymer matrix. For example, in certain embodiments, the components can be encapsulated in, surrounded by, and / or dispersed throughout the polymer matrix. Alternatively or additionally, the components can be bound to one or more polymers of the polymer matrix by hydrophobic interactions, charge interactions, van der Waals forces, and the like. Various polymers and methods for forming polymer matrices are known in the art.

  The polymer can be a natural or non-natural (synthetic) polymer. The polymer can be a homopolymer or a copolymer comprising two or more monomers. With respect to the sequence, the copolymer may be random, block, or may contain a combination of random and block sequences. Usually, the polymer according to the invention is an organic polymer.

  In certain embodiments, the polymer comprises polyester, polycarbonate, polyamide, or polyether, or units thereof. In other embodiments, the polymer is poly (ethylene glycol) (PEG), polypropylene glycol, poly (lactic acid), poly (glycolic acid), poly (lactic acid-co-glycolic acid), or polycaprolactone, or units thereof including. In certain embodiments, the polymer is preferably biodegradable. Thus, in these embodiments, when the polymer comprises a polyether such as poly (ethylene glycol) or polypropylene glycol or units thereof, the polymer is biodegradable with the polyether such that the polymer is biodegradable. It is preferred to include a block copolymer with the polymer. In other embodiments, the polymer does not comprise only polyethers or units thereof, such as poly (ethylene glycol) or polypropylene glycol or units thereof.

  Other examples of polymers suitable for use in the present invention include, but are not limited to, polyethylene, polycarbonate (eg, poly (1,3-dioxane-2one)), polyanhydrides (eg, poly (sebacin) Acid anhydride)), polypropyl fumarate, polyamide (eg, polycaprolactam), polyacetal, polyether, polyester (eg, polylactide, polyglycolide, polylactide-co-glycolide, polycaprolactone, polyhydroxyacid) (eg, polyhydroxyacid) (Β-hydroxyalkanoate))), poly (orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polyurea, polystyrene, and poly Amine, polylysine, polylysine -PEG copolymers, and poly (ethylene imine), poly (ethylene imine) -PEG copolymers.

  In certain embodiments, the polymer according to the present invention includes polyesters (eg, polylactic acid, poly (lactic acid-co-glycolic acid), polycaprolactone, polyvalerolactone, poly (1,3-dioxane-2one)); Polyanhydrides (eg, poly (sebacic anhydride)); polyethers (eg, polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; C. F. R.) § 177.2600 includes polymers approved for human use by the US Food and Drug Administration (FDA).

  In certain embodiments, the polymer can be hydrophilic. For example, the polymer has an anionic group (eg, phosphate group, sulfate group, carboxylic acid group); a cationic group (eg, quaternary amine group); or a polar group (eg, hydroxyl group, thiol group, amine group). ). In certain embodiments, a synthetic nanocarrier comprising a hydrophilic polymer matrix creates a hydrophilic environment within the synthetic nanocarrier. In certain embodiments, the polymer can be hydrophobic. In certain embodiments, a synthetic nanocarrier comprising a hydrophobic polymer matrix creates a hydrophobic environment within the synthetic nanocarrier. The choice of hydrophilicity or hydrophobicity of the polymer can affect the nature of the material that is incorporated (eg, bound) within the synthetic nanocarrier.

  In certain embodiments, the polymer can be modified with one or more moieties and / or functional groups. Various moieties or functional groups can be used in accordance with the present invention. In certain embodiments, the polymer can be modified with acrylic polyacetals derived from polyethylene glycol (PEG), carbohydrates, and / or polysaccharides (Papisov, 2001, ACS Symposium Series, 786: 301). Certain embodiments are described in Gref et al. U.S. Pat. No. 5,543,158, or Von Andrian et al. Can be made using the general teachings of WO 2009/051837 of International Publication No.

  In certain embodiments, the polymer can be modified with lipid or fatty acid groups. In certain embodiments, the fatty acid group can be one or more of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, or lignoceric acid. . In certain embodiments, the fatty acid group is palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, α-linoleic acid, γ-linoleic acid, arachidonic acid, gadoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or erucic acid. Can be one or more.

  In certain embodiments, the polymer is a copolymer comprising lactic and glycolic acid units, such as poly (lactic acid-co-glycolic acid) and poly (lactide-co-glycolide), collectively referred to herein as “PLGA”; And a glycolic acid unit referred to herein as “PGA” and a poly-L-lactic acid, poly-D-lactic acid, poly-D, L-lactic acid, poly-L, collectively referred to herein as “PLA”. -Polyesters comprising homopolymers comprising lactic acid units such as lactide, poly-D-lactide and poly-D, L-lactide. In certain embodiments, exemplary polyesters include, for example, polyhydroxy acids; PEG copolymers and copolymers of lactide and glycolide (eg, PLA-PEG copolymer, PGA-PEG copolymer, PLGA-PEG copolymer, and derivatives thereof). In certain embodiments, polyesters include, for example, poly (caprolactone), poly (caprolactone) -PEG copolymer, poly (L-lactide-co-L-lysine), poly (serine ester), poly (4- Hydroxy-L-proline ester), poly [[alpha]-(4-aminobutyl) -L-glycolic acid], and derivatives thereof.

  In certain embodiments, the polymer can be PLGA. PLGA is a biocompatible and biodegradable copolymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid: glycolic acid. The lactic acid can be L-lactic acid, D-lactic acid, or D, L-lactic acid. The degradation rate of PLGA can be adjusted by changing the ratio of lactic acid: glycolic acid. In certain embodiments, the PLGA used in accordance with the present invention is about 85:15, about 75:25, about 60:40, about 50:50, about 40:60, about 25:75, or about 15:85. Characterized by the lactic acid: glycolic acid ratio.

  In certain embodiments, the polymer can be one or more acrylic polymers. In certain embodiments, acrylic polymers include, for example, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly (acrylic acid), poly (methacrylic acid), methacrylic acid. Alkylamide copolymers, poly (methyl methacrylate), poly (methacrylic anhydride), methyl methacrylate, polymethacrylate, poly (methyl methacrylate) copolymer, polyacrylamide, aminoalkyl methacrylate copolymer, glycidyl methacrylate copolymer, polycyanoacrylate, and the above polymers A combination including one or more of the above. The acrylic polymer may comprise a fully polymerized copolymer of acrylic acid and methacrylic acid ester having a low content of quaternary ammonium groups.

  In certain embodiments, the polymer can be a cationic polymer. In general, cationic polymers can condense and / or protect negatively charged strands of nucleic acids (eg, DNA, or derivatives thereof). Amine-containing polymers such as poly (lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6: 7), poly (ethyleneimine) (PEI; Bousif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92: 7297), and poly (amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad). Sci., USA, 93: 4897; Tang et al., 1996, Bioconjugate Chem., 7: 703; and Haensler et al., 1993, Bioconj. gate Chem, 4:. 372) is positively charged at physiological pH, form nucleic acid ion pair mediates transfection in a variety of cell lines. In embodiments, the synthetic nanocarriers of the present invention may not include (ie may exclude) cationic polymers.

  In certain embodiments, the polymer may be a degradable polyester having cationic side chains (Putnam et al., 1999, Macromolecules, 32: 3658; Barrera et al., 1993, J. Am. Chem. Soc., 115: 11010; Kwon et al., 1989, Macromolecules, 22: 3250; Lim et al., 1999, J. Am. Chem. Soc., 121: 5633; and Zhou et al., 1990, Macromolecules, 23: 3399. ). Examples of these polyesters include poly (L-lactide-co-L-lysine) (Barrera et al., 1993, J. Am. Chem. Soc., 115: 11010), poly (serine ester) (Zhou et al., 1990, Macromolecules, 23: 3399), poly (4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32: 3658; and Lim et al., 1999, J. Am. Chem. Soc., 121: 5633), and poly (4-hydroxy-L-proline ester) (Putnam et al., 1999, Macromolecules, 32: 3658; and Lim et al., 1999, J. Am. C. em.Soc, 121:. 5633) can be mentioned.

  The properties of these and other polymers and methods for preparing them are well known in the art (eg, US Pat. No. 6,123,727; US Pat. No. 5,804,178). No. 5,770,417; No. 5,736,372; No. 5,716,404; No. 6,095,148; No. 5 , 837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600 No. 5,399,665; No. 5,019,379; No. 5,010,167; No. 4,806,621; No. 638,045; and No. 4,946,929. Wang et al., 2001, J. Am.Chem.Soc., 123: 9480; Lim et al., 2001, J.Am.Chem.Soc., 123: 2460; Langer, 2000, Acc. Res., 33:94; see Langer, 1999, J. Control. Release, 62: 7; and Uhrich et al., 1999, Chem. Rev., 99: 3181). More generally, various methods for synthesizing certain suitable polymers are described in Concise Encyclopedia of Polymer Science and Polymer Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry Chemistry. , Prentice-Hall, 1981; Deming et al. , 1997, Nature, 390: 386; and US Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818. , 732 specification.

  In certain embodiments, the polymer can be a linear or branched polymer. In certain embodiments, the polymer can be a dendrimer. In certain embodiments, the polymers can be substantially cross-linked to each other. In certain embodiments, the polymer may be substantially free of crosslinking. In certain embodiments, the polymer can be used in accordance with the present invention without a crosslinking step. It should be further understood that the synthetic nanocarriers of the present invention can include block copolymers, graft copolymers, blends, mixtures, and / or adducts of any of the above and other polymers. Those skilled in the art will recognize that the polymers listed herein represent an illustrative rather than a comprehensive list of polymers that can be used in accordance with the present invention.

  The composition according to the present invention comprises a synthetic nanocarrier in combination with a pharmaceutically acceptable excipient such as a preservative, buffer, saline, or phosphate buffered saline. The composition can be made using conventional drug manufacturing and formulation techniques to arrive at useful dosage forms. In one embodiment, the synthetic nanocarriers of the invention are suspended in a sterile saline solution for injection with a preservative.

  In embodiments, when preparing a synthetic nanocarrier as a carrier, methods for attaching the components to the synthetic nanocarrier may be useful. If the component is a small molecule, it may be advantageous to attach the component to the polymer prior to assembly of the synthetic nanocarrier. In an embodiment, the components are coupled to a polymer and then the polymer composite is not used to construct a synthetic nanocarrier, but instead of surface groups (using these surface groups to bind the components to the synthetic nanocarrier. It may also be advantageous to prepare synthetic nanocarriers with) used.

  In certain embodiments, the bond can be a covalent linker. In an embodiment, the peptide according to the invention is obtained by a 1,3-dipolar cycloaddition reaction between an azide group on the surface of the nanocarrier and an antigen or immunosuppressant containing an alkyne group, or on the surface of the nanocarrier. Can be covalently attached to the outer surface via a 1,2,3-triazole linker formed by a 1,3-dipolar cycloaddition reaction of an alkyne with an azide group containing antigen or immunosuppressant. Such a cycloaddition reaction is preferably carried out in the presence of a Cu (I) catalyst together with a suitable Cu (I) ligand and a reducing agent to reduce the Cu (II) compound to a catalytically active Cu (I) compound. Done in This Cu (I) -catalyzed azide-alkyne cycloaddition (CuAAC) is sometimes referred to as a click reaction.

  Further, the covalent bond can include a covalent linker including an amide linker, disulfide linker, thioether linker, hydrazone linker, hydrazide linker, imine or oxime linker, urea or thiourea linker, amidine linker, amine linker, and sulfonamide linker. .

  An amide linker is formed through an amide bond between an amine on one component, such as an antigen or immunosuppressant, and a carboxylic acid group on a second component, such as a nanocarrier. The amide bond in the linker can be made using any of the conventional amide bond forming reactions with suitably protected amino acids and activated carboxylic acids (such as esters activated with N-hydroxysuccinimide).

  A disulfide linker is made, for example, by formation of a disulfide (S—S) bond between two sulfur atoms in the form of R 1 —S—S—R 2. A disulfide bond is a thiol exchange of an antigen or immunosuppressant containing a thiol / mercaptan group (-SH) with another activated thiol group in the polymer or nanocarrier, or of a nanocarrier containing a thiol / mercaptan group. It can be formed by thiol exchange with an antigen or immunosuppressant containing an activated thiol group.

Triazole linker, in particular 1,2,3-triazole of the form
(Wherein R1 and R2 may be any chemicals) are bound to an azide bound to a first component such as a nanocarrier and a second component such as an antigen or immunosuppressant. Prepared by a 1,3-dipolar cycloaddition reaction with a terminal alkyne. The 1,3-dipolar cycloaddition reaction is carried out with or without a catalyst, preferably with a Cu (I) catalyst that joins the two components by 1,2,3-triazole functional groups. This chemistry is described in Sharpless et al. , Angew. Chem. Int. Ed. 41 (14), 2596, (2002) and Meldal, et al, Chem. Rev. 2008, 108 (8), 2952-3015, and is often referred to as the “click” reaction or CuAAC.

  In an embodiment, a polymer containing an azide group or alkyne group at the end of the polymer chain is prepared. This polymer is then used to prepare a synthetic nanocarrier such that multiple alkyne or azide groups are placed on the surface of the corresponding nanocarrier. Alternatively, synthetic nanocarriers can be prepared by another route and then functionalized with alkyne or azide groups. The component is prepared by the presence of either an alkyne group (if the polymer contains an azide group) or an azide group (if the polymer contains an alkyne group). The component is then coupled to the nanocarrier by a 1,3-dipolar cycloaddition reaction with or without a catalyst that covalently bonds the component to the particle via a 1,4-disubstituted 1,2,3-triazole linker. Reacted.

  A thioether linker is made, for example, by the formation of a sulfur-carbon (thioether) bond in the form of R1-S-R2. Thioethers can be made by either alkylating a thiol / mercaptan (—SH) group in one component with an alkylating group such as a halide or epoxide in the second component. A thioether linker can also be formed by Michael addition of a thiol / mercaptan group in one component to an electron-deficient alkene group in a second component that contains a maleimide or vinylsulfone group as a Michael acceptor. In another method, a thioether linker can be prepared by a radical thiol-ene reaction between a thiol / mercaptan group in one component and an alkene group in a second component.

  The hydrazone linker is made by reaction of the hydrazide group in one component with the aldehyde / ketone group in the second component.

  A hydrazide linker is formed by the reaction of a hydrazine group in one component with a carboxylic acid group in a second component. Such a reaction is generally performed using a chemistry similar to the formation of an amide bond in which a carboxylic acid is activated with an activating reagent.

  The imine or oxime linker is formed by reaction of an amine or N-alkoxyamine (or aminooxy) group in one component with an aldehyde or ketone group in the second component.

  A urea or thiourea linker is prepared by reaction of an amine group in one component with an isocyanate or thioisocyanate group in a second component.

  Amidine linkers are prepared by reaction of an amine group in one component with an imide ester group in a second component.

  The amine linker is made by an alkylation reaction between an amine group in one component and an alkylating group such as a halide, epoxide, or sulfonate group in the second component. Alternatively, the amine linker can also be reductive between an amine group in one component and an aldehyde or ketone group in the second component using a suitable reducing reagent such as sodium cyanoborohydride or sodium triacetoxyborohydride. Can be made by amination.

  The sulfonamide linker is made by reaction of an amine group in one component with a sulfonyl halide group (such as sulfonyl chloride) in the second component.

  The sulfone linker is made by Michael addition of a nucleophile to vinyl sulfone. Either vinyl sulfone or nucleophile can be on the surface of the nanocarrier or bound to a component.

  The component can also be bound to the nanocarrier by non-covalent methods. For example, a negatively charged antigen or immunosuppressant can be bound to a positively charged nanocarrier by electrostatic adsorption. The component containing the metal ligand can also be bound to the nanocarrier containing the metal complex via the metal ligand complex.

  In embodiments, the component can be coupled to a polymer, such as polylactic acid-block-polyethylene glycol, prior to assembly of the synthetic nanocarrier, or the synthetic nanocarrier can have reactive or activatable groups on its surface. Can be formed using. In the latter case, the components can be prepared with groups that are compatible with the binding chemistry presented by the surface of the synthetic nanocarrier. In other embodiments, the peptide component can be attached to the VLP or liposome using a suitable linker. A linker is a compound or reagent capable of linking two molecules together. In one embodiment, the linker can be a homobifunctional or heterobifunctional reagent, as described in Hermanson 2008. For example, a VLP or liposome synthetic nanocarrier containing a carboxylic acid group on the surface is treated with a homobifunctional linker, adipic acid dihydrazide (ADH) in the presence of EDC, and the corresponding synthetic nanocarrier is converted by the ADH linker. Can be formed. The resulting ADH-bonded synthetic nanocarrier is then combined with the peptide component containing an acidic group via the other end of the ADH linker in NC to produce the corresponding VLP or liposome peptide complex.

  For a detailed description of available binding methods, see Hermanson GT “Bioconjugate Techniques”, 2nd Edition Published by Academic Press, Inc. , 2008. In addition to covalent bonding, the components can be bound by adsorption to preformed synthetic nanocarriers, or the components can be bound by encapsulation during formation of the synthetic nanocarriers.

  Any immunosuppressive agent provided herein can be bound to a synthetic nanocarrier. Immunosuppressive agents include but are not limited to statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase (HDAC) inhibitors; Corticosteroids; inhibitors of mitochondrial function such as rotenone; P38 inhibitors; NF-κβ inhibitors; adenosine receptor agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors such as phosphodiesterase 4 inhibitors; proteasome inhibitors; G protein-coupled receptor agonist; G protein-coupled receptor antagonist; Glucocorticoid; Retinoid; Cytokine inhibitor; Cytokine receptor inhibitor; Cytokine receptor activator; Peroxisome proliferator activation Receptor antagonists; peroxisome proliferator-activated receptor agonist; histone deacetylase inhibitor; calcineurin inhibitor; phosphatase inhibitors, and oxidation ATP and the like. Examples of immunosuppressants include IDO, vitamin D3, cyclosporin A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine, 6-mercaptopurine, aspirin, niflumic acid, estriol, triptolide, interleukins (for example, IL- 1, IL-10), cyclosporin A, siRNA targeted cytokines or cytokine receptors.

  Examples of statins include atorvastatin (LIPITOR (R), TORVAST (R)), cerivastatin, fluvastatin (LESCOL (R), LESCOL (R) XL), lovastatin (MEVACOR (R), ALTOCOR ( (Registered trademark), ALTOPREV (registered trademark), mevastatin (COMPACTIN (registered trademark)), pitavastatin (LIVALO (registered trademark), PIAVA (registered trademark)), rosuvastatin (PRACHAROL (registered trademark), SELEKTINE (registered trademark), LIPOSSTAT) (Registered trademark)), rosuvastatin (CRESTOR (registered trademark)), and simvastatin (ZOCOR (registered trademark), LIPEX (registered trademark)).

  Examples of mTOR inhibitors include rapamycin and analogs thereof (eg, CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap), C16- (S) -butylsulfonamido rapamycin (C16-BSrap) , C16- (S) -3-methylindole rapamycin (C16-iRap) (Bayl et al. Chemistry & Biology 2006, 13: 99-107)), AZD8055, BEZ235 (NVP-BEZ235), chrysophate acid Nord), deforolimus (MK-8669), everolimus (RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (Selleck (Hou (Ston, TX, USA)).

  Examples of TGF-β signaling agents include TGF-β ligands (eg, activin A, GDF1, GDF11, bone morphogenetic proteins, nodal, TGF-βs) and their receptors (eg, ACVR1B, ACVR1C, ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B, TGFβRI, TGFβRII), R-SMADS / co-SMADS (eg, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD8), and ligand inhibitors (eg, holistatin, noggin, chodin, DAN) , Lefty, LTBP1, THBS1, and decorin).

  Examples of inhibitors of mitochondrial function include atractyloside (dipotassium salt), boncrecynic acid (triammonium salt), carbonylcyanide m-chlorophenylhydrazone, carboxy atractyloside (eg, Attractylis gummifera) Origin), CGP-37157, (−)-deguelin (eg, from Mundulea sericea), F16, hexokinase II VDAC binding domain peptide, oligomycin, rotenone, Ru360, SFK1, and valinomycin (eg, streptomycin) And C. fluvissimus (EMD4Biosciences, USA).

  Examples of P38 inhibitors include SB-203580 (4- (4-fluorophenyl) -2- (4-methylsulfinylphenyl) -5- (4-pyridyl) 1H-imidazole), SB-239063 (trans-1 -(4hydroxycyclohexyl) -4- (fluorophenyl) -5- (2-methoxy-pyrimidin-4-yl) imidazole), SB-220025 (5- (2 amino-4-pyrimidinyl) -4- (4- Fluorophenyl) -1- (4-piperidinyl) imidazole)), and ARRY-797.

  Examples of NF (eg, NK-κβ) inhibitors include IFRD1,2- (1,8-naphthyridin-2-yl) -phenol, 5-aminosalicylic acid, BAY 11-7082, BAY 11-7085, CAPE ( Caffeic acid phenethyl ester), diethyl maleate, IKK-2 inhibitor IV, IMD 0354, lactacystin, MG-132 [Z-Leu-Leu-Leu-CHO], NFκB activation inhibitor III, NF-κB activation Inhibitor II, JSH-23, parthenolide, phenylarsine oxide (PAO), PPM-18, pyrrolidinedithiocarbamate ammonium salt, QNZ, RO 106-9920, rocagramid, rocagramid AL, rocagramid C, rocagramid I, rocagramid J, rocagraol, (R) -MG-1 32, sodium salicylate, triptolide (PG490), and wederolactone.

  Examples of adenosine receptor agonists include CGS-21680 and ATL-146e.

  Examples of prostaglandin E2 agonists include E-prostanoid 2 and E-prostanoid 4.

  Examples of phosphodiesterase inhibitors (non-selective and selective inhibitors) include caffeine, aminophylline, IBMX (3-isobutyl-1-methylxanthine), paraxanthine, pentoxyphylline, theobromine, theophylline, methylated xanthine, Vinpocetine, EHNA (erythro-9- (2-hydroxy-3-nonyl) adenine), anagrelide, enoximone (PERFANTM), milrinone, levosimendan, mesembrine, ibudilast, picramilast, luteolin, drotavelin, roflumilast (DAXASTM, DALIDESTM, DALIRESTM, DALIDESTM, DALIDESTM) REVATION (registered trademark), VIAGRA (registered trademark), Tadalafil (ADCIRCA (registered trademark), CIALIS (registered trademark)), Vardena Le (levitra (registered trademark), STAXYN (registered trademark)), udenafil, Avanafil, icariin, 4-methylpiperazine, and include pyrazolopyrimidine -7-1.

  Examples of proteasome inhibitors include bortezomib, disulfiram, epigallocatechin-3-gallate, and salinosporamide A.

  Examples of kinase inhibitors include bevacizumab, BIBW 2992, cetuximab (ERBITUX®), imatinib (GLEEVEC®), trastuzumab (HERCEPTIN®), gefitinib (IRESSA®), ranibizumab (LUCENTIS (registered trademark)), pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib, E7080, pazopanib, mubritinib.

  Examples of glucocorticoids include hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (DOCA), and aldosterone. .

  Examples of retinoids include retinol, retinal, tretinoin (retinoic acid, RETIN-A (registered trademark)), isotretinoin (ACCUTANE (registered trademark), AMMNSTEM (registered trademark), CLARAVIS (registered trademark), SOTRET (registered trademark) ), Alitretinoin (PANRETIN®), etretinate (TEGISON®) and its metabolites acitretin (SORIATENE®), tazarotene (TAZARAC®, AVAGE®, ZORAC®) Trademark)), bexarotene (TARGRETIN®), and adapalene (DIFFERIN®).

  Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonist, IGFBP, TNF-BF, uromodulin, α-2-macroglobulin, cyclosporin A, pentamidine, and pentoxifylline (PENTOPAK®, PENTOXIL®) ), TRENTAL (registered trademark)).

  Examples of peroxisome proliferator-activated receptor antagonists include GW9662, PPARγ antagonist III, G335, T0070907 (EMD4Biosciences, USA).

  Examples of peroxisome proliferator-activated receptor agonists include pioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY 171883, PPARγ activator, Fmoc-Leu, troglitazone, and WY-14463 (EMD4Biosciences) , USA).

  Examples of histone deacetylase inhibitors include hydroxamic acids (or hydroxamates) such as trichostatin A, cyclic tetrapeptides (such as trapoxin B) and depsipeptides, benzamides, electrophilic ketones, phenylbutyrate and valproic acid, etc. Aliphatic acid compounds, hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589), benzamides such as entinostat (MS-275), CI994, and mosetinostat (MGCD0103), nicotinamide , Derivatives of NAD, dihydrocoumarin, naphthopyranone, and 2-hydroxynaphthalaldehyde.

  Examples of calcineurin inhibitors include cyclosporine, pimecrolimus, voclosporine, and tacrolimus.

  Examples of phosphatase inhibitors include BN82002 hydrochloride, CP-91149, caliculin A, cantharidinic acid, cantharidin, cypermethrin, ethyl-3,4-defostatin, fostriecin sodium salt, MAZ51, methyl-3,4- Defostatin, NSC 95397, norcantharidin, okadaic acid ammonium salt, okadaic acid, okadaic acid potassium salt, okadaic acid sodium salt, phenylarsine oxide, various phosphatase inhibitor mixtures, proteins Phosphatase 1C, protein phosphatase 2A inhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2, sodium orthovanadate Can be mentioned.

  In certain embodiments, the APC-presentable antigens described herein are also bound to synthetic nanocarriers. In certain embodiments, APC presentable antigens are bound to the same or different synthetic nanocarriers depending on which immunosuppressive agent is bound. In other embodiments, the APC presentable antigen is not bound to any synthetic nanocarrier. APC presentable antigens include any of the antigens provided herein. Such antigens include inflammatory diseases, autoimmune diseases, allergies, graft-versus-host disease, antigens associated with organ or tissue rejection, transplantation antigens and therapeutic protein antigens.

  Examples of therapeutic proteins include, but are not limited to, infusible therapeutic proteins, enzymes, enzyme cofactors, hormones, blood clotting factors, cytokines and interferons, growth factors, monoclonal antibodies, and polyclonal antibodies (eg, And those that are associated with Pompe disease, such as alglucosidase alpha, rhGAA (eg, Myozyme and Lumizyme (Genzyme)), and therapeutic proteins in the blood coagulation cascade. Examples of therapeutic proteins include, but are not limited to, Factor VIII, Factor VII, Factor IX, Factor V, von Willebrand factor, von Heldebrand factor eblant Factor), tissue plasminogen activator, insulin, growth hormone, erythropoietin alpha, VEGF, thrombopoietin, lysozyme, antithrombin, etc. Therapeutic proteins also include adipokines such as leptin and adiponectin. Other examples are as described below and elsewhere herein, including any fragment or derivative of a therapeutic protein provided as an antigen.

  Examples of therapeutic proteins used for enzyme replacement therapy in subjects suffering from lysosomal storage disorders include, but are not limited to, imiglucerase (eg, Cerezyme ™), Fabry disease for the treatment of Gaucher disease A-galactosidase A (a-gal A) (e.g., agarsidase beta, FABRYZYME (TM)), acid a-glucosidase (GAA) (e.g., alglucosidase alpha, LUMIZYME) for the treatment of Pompe disease (Trademark), MYOZYME (TM), arylsulfatase B for the treatment of mucopolysaccharidosis (eg, laronidase, ALDURAZYME (TM), idursulfase, ELAPASE (TM), arylsulfatase B, NAGLAZYME (TM) And the like.

  Examples of enzymes include oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase.

  Examples of hormones include melatonin (N-acetyl-5-methoxytryptamine), serotonin, thyroxine (or tetraiodothyronine) (thyroid hormone), triiodothyronine (thyroid hormone), epinephrine (or adrenaline), norepinephrine ( Or noradrenaline), dopamine (or prolactin-inhibiting hormone), anti-Muellerian hormone (or Muller-inhibitory factor or hormone), adiponectin, corticotropin (or corticotropin), angiotensinogen and angiotensin, antidiuretic hormone (or vasopressin, Arginine vasopressin), atrial natriuretic peptide (or atriopeptin), calcitonin, cholecystokinin, corticotropin releasing hormone, Lithropoietin, follicle stimulating hormone, gastrin, ghrelin, glucagon, glucagon-like peptide (GLP-1), GIP, gonadotropin releasing hormone, growth hormone releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin Like growth factor (or somatomedin), leptin, luteinizing hormone, melanocyte stimulating hormone, orexin, oxytocin, parathyroid hormone, prolactin, relaxin, secretin, somatostatin, thrombopoietin, thyroid stimulating hormone (or thyrotropin), thyrotropin releasing hormone , Cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, dihydrotestosterone, Tradiol, estrone, estriol, progesterone, calcitriol (1,25-dihydroxyvitamin D3), calcidiol (25-hydroxyvitamin D3), prostaglandin, leukotriene, prostacyclin, thromboxane, prolactin releasing hormone, lipotropin, Examples include brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalin.

  Examples of blood and blood coagulation factors include factor I (fibrinogen), factor II (prothrombin), tissue factor, factor V (proacrelin, unstable factor), factor VII (stable factor, proconvertin) ), Factor VIII (anti-hemophilic globulin), factor IX (Christmas factor or plasma thromboplastin component), factor X (Stuart-Prower factor), factor Xa, factor XI, factor XII (Hageman factor) , Factor XIII (fibrin stabilizing factor), von Willebrand factor, prekallikrein (Fletcher factor), high molecular weight kininogen (HMWK) (Fitzgerald factor), fibronectin, fibrin, thrombin, antithrombin III, heparin cofactor II , Protein C, Protein S, Pro In Z, protein Z-related protease inhibitor (ZPI), plasminogen, α2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1), plus Minogen activator inhibitor-2 (PAI2), cancer-derived procoagulant, and epoetin alfa (Epogen, Procrit).

  Examples of cytokines include lymphokines, interleukins, and chemokines, type 1 cytokines such as IFN-γ, TGF-β, and type 2 cytokines such as IL-4, IL-10, and IL-13.

  Examples of growth factors include adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO) , Fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), growth differentiation factor-9 ( GDF9), hepatocyte growth factor (HGF), hepatocellular carcinoma-derived growth factor (HDGF), insulin-like growth factor (IGF), migration stimulating factor, myostatin (GDF-8), nerve growth factor (NGF) and other nerves Trophic factor, platelet derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor α (T F-α), transforming growth factor β (TGF-β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), Wnt signaling pathway, placental growth factor (PlGF), [(bovine fetus Somatotropin)] (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7.

  Examples of monoclonal antibodies include abagobomab, abciximab, adalimumab, adecatumumab, aferimomab, aftuzumab, alacizumab pegor, ALD, alemtuzumab, pentetate altumomab, anatomomabumafenatox, anthrumumabumab , Acelizumab, atolizumab, tocilizumab, betizumab, betizumab, betizumab, betizumab, betizumab, betizumab Zumab Meltansine, Capromabu Pentide, Katsumaxomab, Cederiz , Sertolizumab Pegor, Cetuximab, Citatuzumab Bogatox, Sixtumumab, Clenoliximab, Cribatuzumab Tetraxetane, Konatumumab, Dacetuzumab, Daclizumab, Daratumumab, Denosumumab, Detumoma , Eculizumab, edabacomab, edrecolumab, efarizumab, eflizumab, eflizumab, eflizumab, erizumabumab , Font lizumab, folavirumab, fresolimumab, galiximab Gantenerumab, gabilimoumab, gemtuzumab ozogamicin, GC1008, gilentuximab, grembatumab vedotin, golimumab, gomilyximab, ivalizumab, ibritumomab Intetumumab, Inolimomab, Inotuzumab, Ozogamicin, Ipirimumab, Iritumumab, Keriximab, Rabetuzumab, Lebrizumab, Remaresomab, Relderimumab, Lexatumumab, Ribibumabumabumab, Roltuzumab , Metelimumab, miratuzumab, minletumomab, mi Momab, moroliumab, motavizumab, muromonumab-CD3, nacoloma butafenatox, naptomomab estafenatox, natalizumab, nevacumab, nesitumumab, nerellimomab, nimotuzumab, nofetumab momapentane, ocrelizumab Ruth's Mabu Mona Toku vinegar, oregovomab, Oterikishizumabu, Pagibakishimabu, palivizumab, panitumumab, Panobakumabu, Pasukorizumabu, pemtumomab, pertuzumab, pexelizumab, Pintsumomabu, Puririkishimabu, Puritsumumabu, Rafibirumabu, Ramucirumab, ranibizumab, Rakishibakumabu, Regabirumabu, Resurizumabu, Rirotsumumabu, rituximab, Robatsumumabu, Rontalizumab, Roverizumab, Prizumab, Satsumomab pendetide, Sebirumab, Cibrotuzumab, Cifalimumab, Siltuximab, Ciprizumab, Soranezumab, Sonepizumab, Sontuzumab, Stamlumumab, Slesomab, Tacuzumab Tetrazetane, Tadozumab, Tadozumab Mabuaritox, Tenatumomab, Teneriximab, Teplizumab, Ticilimumab (Tremelimumab), Tigatuzumab, Tocilizumab (Atrizumab), Tralizumab, Tositumumab, Trastuzumab, Tremelimumab, Stecozumab Celmoumab Vizilizumab, boroximab, botumumab, the Rutumumab, zanolimumab, dilarimumab, and zolimoumab ritox.

  Examples of therapeutic proteins for infusion therapy or injection include, for example, tocilizumab (Roche / Actemra®), α-1 antitrypsin (Kamada / AAT), Hematide® (Affymax and Takeda) ), Synthetic peptides), albuinterferon α-2b (Novatis / Zalbin ™), Rhucin® (Pharming Group, C1 inhibitor replacement therapy), tesamorelin (Theratechnologies / Egrifta, synthetic growth hormone releasing factor), ocrelizumab (Genentech, Roche and Biogen), belimumab (GlaxoSmithKline / Benlysta®), pegloticase (Sav ent Pharmaceuticals / Krystexxa (TM)), Tari guru seller zero alpha (Protalix / Uplyso), go up mannosidase alpha (Shire / Replagal (TM)), Bella guru seller zero alpha (Shire), and the like.

  Additional therapeutic proteins useful according to embodiments of the present invention will be apparent to those skilled in the art and the present invention is not limited in this regard.

  In certain embodiments, components such as antigens or immunosuppressive agents can be isolated. “Isolated” refers to the element being present in an amount sufficient to be separated from its natural environment and to allow its identification or use. This means, for example, that the element can be (i) selectively produced by expression cloning or (ii) purified by chromatography or electrophoresis. An isolated element can be substantially pure, but need not be substantially pure. Since the isolated element can be mixed with pharmaceutically acceptable excipients in a pharmaceutical formulation, the element can account for only a small percentage by weight of the formulation. Nevertheless, an element is isolated in that it has been separated from a substance that may be associated with it in a biological system, i.e. isolated from other lipids or proteins. Any of the elements provided herein may be isolated. Any of the antigens provided herein can be included in the composition in isolated form.

D. Methods for Making and Using the Compositions of the Invention and Related Methods Synthetic nanocarriers can be prepared using a variety of methods known in the art. For example, synthetic nanocarriers include nanoprecipitation, flow focusing using fluid channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion methods, microemulsion methods, It can be formed by methods such as processing, nanofabrication, sacrificial layers, simple coacervation and complex coacervation, and other methods well known to those skilled in the art. Alternatively or additionally, aqueous and organic solvent synthesis for monodisperse semiconductor nanomaterials, conductive nanomaterials, magnetic nanomaterials, organic nanomaterials, and other nanomaterials has been described (Pellegrino et al. , 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat.Sci., 30: 545; and Trinade et al., 2001, Chem. Mat., 13: 3843). Additional methods are described in the literature (see, eg, Doubrow, Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987; 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6: 275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35: 755; U.S. Pat. Nos. 5,578,325 and 6,0078,845. Description: P. Paulicelli et al., “Surface-modified PLGA-base” Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles "Nanomedicine.5 (6): 843-853 see (2010)).

  Various materials include C.I. Astete et al. , “Synthesis and characteristics of PLGA nanoparticles” J. Biometer. Sci. Polymer Edn, Vol. 17, no. 3, pp. 247-289 (2006); Avgoustakis “Pegylated Poly (Lactide) and Poly (Lactide-Co-Glycolide) Nanoparticulates: Preparation, Properties and Possible Applications in Drug3. Reis et al. , “Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticulates” Nanomedicine 2: 8-21 (2006); Paulicelli et al. "Surface-modified PLGA-based Nanoparticulates that can effectively associate and deliver virus-like particles" Nanomedicine. 5 (6): 843-853 (2010) can be encapsulated into synthetic nanocarriers as desired using various methods including but not limited to. Other suitable for encapsulating materials in synthetic nanocarriers, including but not limited to the methods disclosed in US Pat. No. 6,632,671 issued to Unger on October 14, 2003 The method can be used.

  In certain embodiments, synthetic nanocarriers are prepared by a nanoprecipitation process or spray drying. The conditions used to prepare the synthetic nanocarrier can be altered to obtain particles of the desired size or properties (eg, hydrophobic, hydrophilic, external form, “sticky”, shape, etc.). The method of preparing the synthetic nanocarrier and the conditions used (eg, solvent, temperature, concentration, air flow rate, etc.) may depend on the composition of the material and / or polymer matrix that is bound to the synthetic nanocarrier.

  If the particles prepared by any of the above methods have a size range that is outside the desired range, the particles can be sized using, for example, a sieve.

  The components (ie, elements) of the synthetic nanocarrier of the present invention (parts that constitute an immunofeature surface, target moiety, polymer matrix, antigen, immunosuppressant, etc.) are shared by the entire synthetic nanocarrier, eg, one or more They may be joined by a bond or may be joined by one or more linkers. Additional methods for functionalizing synthetic nanocarriers are described in Saltzman et al. U.S. Patent Application Publication No. 2006/0002852, DeSimon et al. U.S. Patent Application Publication No. 2009/0028910, or Murthy et al. From published international patent application WO / 2008/127532 A1.

  Alternatively or additionally, synthetic nanocarriers can be coupled to the elements provided herein, either directly or indirectly through non-covalent interactions. In non-covalent embodiments, non-covalent bonds include charge interactions, affinity interactions, metal coordination, physisorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, There are non-covalent interactions, including but not limited to van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-to-dipole interactions, and / or combinations thereof. Such bonds can be arranged to be on the outer or inner surface of the synthetic nanocarrier of the present invention. In embodiments, encapsulation and / or absorption is in the form of binding. In embodiments, the synthetic nanocarriers of the invention can be combined with an antigen by mixing in the same vehicle or delivery system.

  The population of synthetic nanocarriers can be combined to form a pharmaceutical dosage form according to the present invention using conventional drug mixing methods. These include liquid-liquid mixing where two or more suspensions, each containing one or more subsets of nanocarriers, are combined directly or combined by one or more containers containing a diluent. Also, when synthetic nanocarriers can be manufactured or stored in powder form, dry mixing of the powders can be performed such that resuspension of two or more powders can be performed in a common medium. Depending on the properties of the nanocarriers and their interaction potential, there can be advantages conferred on one or another mixing path.

  Exemplary inventive compositions comprising synthetic nanocarriers include inorganic or organic buffers (eg, phosphate, carbonate, acetate, or citrate sodium or potassium salts) and pH adjusters (eg, Hydrochloric acid, sodium hydroxide or potassium hydroxide, citric acid or acetic acid salts, amino acids and salts thereof) antioxidants (eg ascorbic acid, α-tocopherol), surfactants (eg polysorbate 20, polysorbate 80, poly Oxyethylene 9-10 nonylphenol, sodium desoxycholate), solutions and / or freeze / dissolve stabilizers (eg sucrose, lactose, mannitol, trehalose), osmotic regulators (eg salts or sugars), antibacterial agents (eg Benzoic acid, phenol, gentamicin), antifoam (e.g. Redimethylsiloxane), preservatives (eg thimerosal, 2-phenoxyethanol, EDTA), polymer stabilizers and viscosity modifiers (eg polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and cosolvents (eg glyceryl, polyethylene glycol, Ethanol).

  The composition according to the present invention comprises the synthetic nanocarrier of the present invention in combination with a pharmaceutically acceptable excipient. The composition can be made using conventional drug manufacturing and formulation techniques to arrive at useful dosage forms. Techniques suitable for use in practicing the present invention include Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. et al. Atiemo-Obeng, and Suzanne M .; Kresta, 2004 John Wiley & Sons, Inc. And Pharmaceuticals: The Science of Dosage Form Design, 2nd Ed. Edited by M.M. E. Auten, 2001, Churchill Livingstone. In one embodiment, the synthetic nanocarriers of the invention are suspended in a sterile saline solution for injection with a preservative.

  It should be understood that the compositions of the present invention can be made in any suitable manner and that the present invention is in no way limited to compositions that can be made using the methods described herein. Selection of an appropriate method may require consideration of the characteristics of the particular part involved.

  In certain embodiments, the synthetic nanocarriers of the present invention are manufactured under sterile conditions or ultimately sterilized. This ensures that the resulting composition can be made sterile and non-infectious, thus improving safety when compared to non-sterile compositions. This provides a valuable safety measure, especially if the subject to whom the synthetic nanocarrier is administered has an immunodeficiency, is suffering from an infection, and / or is susceptible to an infection. In certain embodiments, the synthetic nanocarriers of the invention can be lyophilized and stored over a long period of time, in suspension or as a lyophilized powder, depending on the formulation procedure, without loss of activity.

  The composition of the present invention is subcutaneous, intranasal, oral, intravenous, intraperitoneal, intramuscular, transmucosal, transmucosal, sublingual, rectal, eye drop, transpulmonary, intradermal, transdermal, transdermal. (Transcutaneous) or can be administered by various routes, including but not limited to intradermal, or by a combination of these routes. The route of administration also includes administration by inhalation or transpulmonary aerosol. Techniques for preparing aerosol delivery systems are well known to those skilled in the art (see, eg, Scirara and Cutie, “Aerosols,” in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp. 1694, incorporated by reference. 1712).

  An implantable graft or therapeutic protein provided as a cell-based therapy of the present invention may be administered parenterally, intraarterially, intranasally or intravenously, or by injection into a lymph node or anterior chamber, or It can be administered by local administration to the organ or tissue of interest. Administration can be by subcutaneous, intrathecal, intraventricular, intramuscular, intraperitoneal, intracoronary, intrapancreatic, intrahepatic or intrabronchial injection.

  The compositions of the invention can be administered in an effective amount, such as an effective amount described elsewhere herein. The dosage of the dosage form contains different amounts of the synthetic nanocarrier population and / or different amounts of immunosuppressive agents and / or antigens according to the present invention. The amount of synthetic nanocarrier and / or immunosuppressive agent and / or antigen present in the dosage form of the present invention depends on the nature of the antigen and / or immunosuppressive agent, the therapeutic effect achieved, and other such parameters. It can be changed accordingly. In embodiments, a dosage determination test can be performed to determine the optimal therapeutic amount of the population of synthetic nanocarriers and the amount of immunosuppressive agent and / or antigen present in the dosage form. In embodiments, the synthetic nanocarrier and / or immunosuppressive agent and / or antigen is present in the dosage form in an amount effective to generate a tolerogenic immune response to the antigen upon administration to a subject. . It may be possible to determine the amount of immunosuppressive agent and / or antigen effective to produce a tolerogenic immune response using conventional dosage determination tests and techniques in a subject. The dosage forms of the invention can be administered at various frequencies. In preferred embodiments, at least one administration of the dosage form is sufficient to produce a pharmacologically relevant response. In more preferred embodiments, at least 2 doses, at least 3 doses, or at least 4 doses of the dosage form are used to ensure a pharmacologically relevant response.

  Prophylactic administration of the compositions of the invention can be initiated before the onset of the disease, disorder or condition, or therapeutic administration can be initiated after the disease, disorder or condition has established.

  In certain embodiments, administration of the synthetic nanocarrier occurs, for example, prior to administration of a therapeutic protein, an implantable implant, or exposure to an allergen. In exemplary embodiments, synthetic nanocarriers include, but are not limited to, 30, 25, 20, 15, 14, 13, 12, 11, administration of therapeutic proteins, implantable implants or administration to allergens. It is administered one or more times, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 days before. In addition or alternatively, the synthetic nanocarrier can be administered to a subject after administration of a therapeutic protein, an implantable implant, or exposure to an allergen. In exemplary embodiments, synthetic nanocarriers include, but are not limited to, 1, 2, 3, 4, 5, 6, 7, 8 of therapeutic protein, implantable implant administration, or exposure to allergens. , 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 days later, etc.

  In certain embodiments, a maintenance dose (eg, of a synthetic nanocarrier provided herein) is an undesirable immunity in a subject, eg, to maintain a tolerogenic effect obtained after an initial dose. In order to prevent a response or to prevent a subject from becoming a subject at risk of developing an undesirable or undesired level of immune response, initial administration may induce a tolerogenic response in the subject. Once administered, it is administered to the subject. In certain embodiments, the maintenance dose is the same dose as the initial dose administered to the subject. In certain embodiments, the maintenance dose is a lower dose than the initial dose. For example, in certain embodiments, the maintenance dose is about 3/4, about 2/3, about 1/2, about 1/3, about 1/4, about 1/8, about 1/10 of the initial dosage. About 1/20, about 1/25, about 1/50, about 1/100, about 1/1000, about 1 / 10,000, about 1 / 100,000, or about 1/1000, 000 (weight / weight).

  The compositions and methods described herein induce or promote a tolerogenic immune response and / or suppress, modulate, direct or direct an immune response that is undesirable for immunosuppression. Can be used to redirect. The compositions and methods described herein can be used in the diagnosis, prevention and / or treatment of a disease, disorder or condition where immunosuppression (eg, an immune response that produces immune tolerance) would have a therapeutic effect. . Such diseases, disorders or conditions include autoimmune diseases, inflammatory diseases, allergies, organ or tissue rejection and graft versus host disease. The compositions and methods described herein can also be used on subjects who have or will be transplanted. The compositions and methods described herein are administered, are or will be administered a therapeutic protein in which a subject has generated or is expected to generate an undesirable immune response. It can also be used for certain subjects.

  Autoimmune diseases include, but are not limited to, rheumatoid arthritis, multiple sclerosis, immune-mediated or type 1 diabetes, inflammatory bowel disease (eg, Crohn's disease or ulcerative colitis), systemic lupus erythematosus, psoriasis , Scleroderma, autoimmune thyroid disease, alopecia areata, Graves' disease, Guillain-Barre syndrome, celiac disease, Sjogren's syndrome, rheumatic fever, gastritis, autoimmune atrophic gastritis, autoimmune hepatitis, isletitis, ovary Inflammation, testitis, uveitis, lens-induced uveitis, myasthenia gravis, primary myxedema, pernicious anemia, autoimmune hemolytic anemia, Addison's disease, scleroderma, Goodpascher syndrome, nephritis, for example , Glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid, sympathetic ophthalmitis, idiopathic thrombocytopenic purpura, idiopathic leukopenia, Wegener's granulomas and multiple / skin Myositis, and the like.

  Some additional exemplary autoimmune diseases, associated autoantigens, and autoantibodies contemplated for use in the present invention are set forth in Table 1 below:

  Inflammatory diseases include but are not limited to Alzheimer's disease, arthritis, asthma, atherosclerosis, Crohn's disease, colitis, cystic fibrosis, dermatitis, diverticulitis, hepatitis, irritable bowel syndrome ( IBS), lupus erythematosus, muscular dystrophy, nephritis, Parkinson's disease, shingles and ulcerative colitis. Inflammatory diseases include, for example, cardiovascular disease, chronic obstructive pulmonary disease (COPD), bronchiectasis, chronic cholecystitis, tuberculosis, Hashimoto thyroiditis, sepsis, sarcoidosis, silicosis and other pneumoconiosis, and wounds Examples include, but are not limited to, embedded foreign objects. As used herein, the term “sepsis” refers to a well-recognized clinical syndrome associated with the host's systemic inflammatory response to microbial invasion. As used herein, the term “sepsis” refers to a condition usually indicated by fever or hypothermia, tachycardia, and tachypnea, and in severe cases, decreases in blood pressure, organ dysfunction, and even It can even lead to death.

  In certain embodiments, the inflammatory disease is by non-autoimmune inflammatory bowel disease, postoperative adhesion, coronary artery disease, liver fibrosis, acute respiratory distress syndrome, acute inflammatory pancreatitis, endoscopic retrograde cholangiopancreatography Induced pancreatitis, burns, coronary, cerebral and peripheral artery atherogenesis, appendicitis, cholecystitis, diverticulitis, visceral fibrotic disorder, wound healing, skin scarring disorder (keloid, Sweaty abscess), granulomatous disease (sarcoidosis, primary biliary cirrhosis), asthma, pyoderma gangrenosum, sweet syndrome, Behcet's disease, primary sclerosing cholangitis or abscess. In certain preferred embodiments, the inflammatory disease is inflammatory bowel disease (eg, Crohn's disease or ulcerative colitis).

  Inflammatory diseases include but are not limited to Alzheimer's disease, ankylosing spondylitis, arthritis, asthma, atherosclerosis, Behcet's disease, chronic inflammatory demyelinating polyneuritis, Crohn's disease, autoimmunity Inflammatory bowel disease, insulin-dependent diabetes mellitus, diabetes, juvenile diabetes, spontaneous autoimmune diabetes, gastritis, autoimmune atrophic gastritis, autoimmune hepatitis, thyroiditis, Hashimoto's thyroiditis, isletitis, ovitis, Testitis, uveitis, lens-induced uveitis, multiple sclerosis, myasthenia gravis, primary myxedema, hyperthyroidism, pernicious anemia, autoimmune hemolytic anemia, Addison's disease, ankylosing spine Inflammation, sarcoidosis, scleroderma, Goodpasture syndrome, Guillain-Barre syndrome, Graves' disease, glomerulonephritis, psoriasis, pemphigus vulgaris, pemphigoid, blood prolapse, bullous pemphigoid Sympathetic ophthalmitis, idiopathic thrombocytopenic purpura, idiopathic leukopenia, Sjogren's syndrome, systemic sclerosis, Wegener's granulomas, multiple / dermatomyositis, primary biliary cirrhosis, primary sclerosing cholangitis, Lupus or systemic lupus erythematosus.

  Graft-versus-host disease (GVHD) is a complication that can occur after transplantation of pluripotent cells (eg, stem cells) or bone marrow, where newly transplanted material attacks the body of the transplant recipient. In some cases, GVHD occurs after blood transfusion. Graft-versus-host disease can be classified into acute and chronic types. The acute or fulminant form of the disease (aGVHD) is usually observed within the first 100 days after transplantation and is a major transplant challenge due to the morbidity and mortality associated therewith. The chronic form of graft-versus-host disease (cGVHD) usually occurs after 100 days. The development of moderate to severe cases of cGVHD adversely affects long-term survival.

Example 1: Mesoporous silica nanoparticles bound with ibuprofen (theoretical experimental example)
A mesoporous SiO2 nanoparticle core is produced by a sol-gel method. Hexadecyltrimethylammonium bromide (CTAB) (0.5 g) is dissolved in deionized water (500 mL) and then 2 M aqueous NaOH (3.5 mL) is added to the CTAB solution. The solution is stirred for 30 minutes and then tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. The resulting gel is stirred at a temperature of 80 ° C. for 3 hours. The resulting white precipitate is captured by filtration, then washed with deionized water and dried at room temperature. The remaining surfactant is then extracted from the particles by suspending in HCl in ethanol overnight. The particles are washed with ethanol, centrifuged and redispersed by sonication. Repeat this washing procedure two more times.

  Next, the SiO2 nanoparticles are functionalized with amino groups using (3-aminopropyl) -triethoxysilane (APTMS). To do this, the particles are suspended in ethanol (30 mL) and APTMS (50 μL) is added to the suspension. The suspension is left at room temperature for 2 hours, then boiled for 4 hours and the volume is kept constant by adding ethanol periodically. Remaining reactants are removed by five cycles of washing by centrifugation and redispersion in pure ethanol.

  In the separation reaction, a gold seed having a diameter of 1 to 4 nm is generated. All water used in this reaction is first deionized and then distilled from the glass. Add water (45.5 mL) to a 100 mL round bottom flask. While stirring, a 0.2 M aqueous NaOH solution (1.5 mL) is added, followed by a 1% aqueous solution (1.0 mL) of tetrakis (hydroxymethyl) phosphonium chloride (THPC). Two minutes after the addition of the THPC solution, a 10 mg / mL aqueous solution (2 mL) of chloroauric acid aged for at least 15 minutes is added. The gold seed is purified by dialysis against water.

  To form the core-shell nanocarrier, the amino-functionalized SiO2 nanoparticles formed above are first mixed with a gold seed at room temperature for 2 hours. The gold-decorated SiO2 particles are collected by centrifugation and mixed with an aqueous solution of chloroauric acid and potassium bicarbonate to form a gold shell. The particles are then washed by centrifugation and redispersed in water. The ibuprofen is loaded by suspending the particles in a solution of sodium ibuprofen (1 mg / L) for 72 hours. The free ibuprofen is then washed from the particles by centrifugation and redispersed in water.

Example 2: Liposomes containing cyclosporin A (theoretical experimental example)
Liposomes are formed using thin film hydration. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol), cholesterol (32 μmol), and cyclosporin A (6.4 μmol) are dissolved in pure chloroform (3 mL). This lipid solution is added to a 50 mL round bottom flask and the solvent is evaporated in a rotary evaporator at a temperature of 60 ° C. The flask is then flushed with nitrogen gas to remove the remaining solvent. Phosphate buffered saline (2 mL) and 5 glass beads are added to the flask and the lipid membrane is hydrated by shaking at 60 ° C. for 1 hour to form a suspension. The suspension is transferred to a small pressure tube and sonicated at 60 ° C. over 4 cycles of 30 second pulses with a 30 second delay between each pulse. The suspension is then left at room temperature for 2 hours to fully hydrate. Liposomes are washed by centrifugation and then resuspended in fresh phosphate buffered saline.

Example 3: Polymer nanocarrier containing polymer-rapamycin conjugate (theoretical experimental example)
Preparation of PLGA-rapamycin conjugate:
PLGA polymer with acidic end groups (7525 DLG1A, acid number 0.46 mmol / g, Lakeshore Biomaterials; 5 g, 2.3 mmol, 1.0 equiv) is dissolved in 30 mL of dichloromethane (DCM). N, N-dicyclohexylcarbodiimide (1.2 eq, 2.8 mmol, 0.57 g) was added followed by rapamycin (1.0 eq, 2.3 mmol, 2.1 g) and 4-dimethylaminopyridine (DMAP) ( 2.0 equivalents, 4.6 mmol, 0.56 g). The mixture is stirred at room temperature for 2 days. The mixture is then filtered to remove insoluble dicyclohexylurea. The filtrate is concentrated to a volume of approximately 10 mL and added to 100 mL of isopropyl alcohol (IPA) to precipitate the PLGA-rapamycin conjugate. The IPA layer is removed and the polymer is then washed with 50 mL IPA and 50 mL methyl t-butyl ether (MTBE). The polymer is then dried under vacuum at 35 C for 2 days to give PLGA-rapamycin (about 6.5 g) as a white solid.

Preparation of nanocarriers containing PLGA-rapamycin conjugate and ovalbumin peptide (323-339):
Nanocarriers containing PLGA-rapamycin are prepared according to the procedure described in Example 1 as follows:

A solution for nanocarrier formation is prepared as follows:
Solution 1: 20 mg / mL ovalbumin peptide 323-339 in dilute aqueous hydrochloric acid. A solution is prepared by dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution at room temperature. Solution 2: 100 mg / mL PLGA-rapamycin in methylene chloride. A solution is prepared by dissolving LGA-rapamycin in pure methylene chloride. Solution 3: 100 mg / mL PLA-PEG in methylene chloride. A solution is prepared by dissolving PLA-PEG in pure methylene chloride. Solution 4: 50 mg / mL polyvinyl alcohol in 100 mM pH 8 phosphate buffer.

  A first water-in-oil emulsion is first prepared. Solution 1 (0.2 mL), Solution 2 (0.75 mL), and Solution 3 (0.25 mL) are combined in a small pressure tube using a Branson Digital Sonifier 250 at 50% amplitude for 40 seconds. Prepare W1 / O1 by decomposing with ultrasound. Next, Solution 4 (3.0 mL) is combined with the first W1 / O1 emulsion, vortexed for 10 seconds, and sonicated with a Branson Digital Sonifier 250 for 60 seconds at 30% amplitude. To prepare a second emulsion (W1 / O1 / W2). The W1 / O1 / W2 emulsion is added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate methylene chloride and form nanocarriers. Transfer the nanocarrier suspension to a centrifuge tube, centrifuge at 75,600 × g and 4 ° C. for 35 minutes, remove the supernatant, and resuspend the pellet in phosphate buffered saline. Wash part of the carrier. The washing procedure is repeated and the pellet is resuspended in phosphate buffered saline for a final nanocarrier dispersion of about 10 mg / mL.

Example 4: Preparation of gold nanocarrier (AuNC) containing rapamycin (example of theoretical experiment)
Preparation of HS-PEG-rapamycin:
A solution of PEG acid disulfide (1.0 eq), rapamycin (2.0-2.5 eq), DCC (2.5 eq) and DMAP (3.0 eq) in dry DMF was stirred overnight at room temperature. To do. Insoluble dicyclohexylurea is removed by filtration and the filtrate is added to isopropyl alcohol (IPA) to precipitate PEG-disulfide-di-rapamycin ester, washed with IPA and dried. The polymer is then treated with tris (2-carboxyethyl) phosphine hydrochloride in DMF to reduce the PEG disulfide to the thiol PEG rapamycin ester (HS-PEG-rapamycin). The resulting polymer is recovered by precipitation from IPA, dried as described above, and analyzed by H NMR and GPC.

Formation of gold NC (AuNC):
500 mL of an aqueous solution of 1 mM HAuCl 4 is heated to reflux for 10 minutes with vigorous stirring in a 1 L round bottom flask equipped with a condenser. Next, 50 mL of 40 mM trisodium citrate solution is quickly added to the stirring solution. The resulting dark wine red solution is kept at reflux for 25-30 minutes, the heat is removed and the solution is cooled to room temperature. The solution is then filtered through a 0.8 μm membrane filter to obtain an AuNC solution. AuNC is characterized using visible spectroscopy and transmission electron microscopy. AuNC is about 20 nm in diameter, capped with citrate and has a peak absorption at 520 nm.

AuNC conjugate with HS-PEG-rapamycin:
A solution of 150 μl HS-PEG-rapamycin (10 μM in 10 mM pH 9.0 carbonate buffer) was added to 1 mL of 20 nm diameter citrate-capped gold nanocarrier (1.16 nM) and 2500 A molar ratio of thiol to gold of 1 is obtained. The mixture is stirred at room temperature for 1 hour under argon to allow complete exchange of thiol and citrate on the gold nanocarrier. Next, AuNC complexed with PEG-rapamycin on the surface is purified by centrifugation at 12,000 g for 30 minutes. The supernatant is decanted and the pellet containing AuNC-S-PEG-rapamycin is then washed with 1 × PBS buffer. The purified gold-PEG-rapamycin nanocarrier is then resuspended in a suitable buffer for further analysis and bioassay.

Example 5: Synthetic nanocarrier immune response material with bound rapamycin with and without ovalbumin peptide (323-339) 17 amino acid peptide known to be T and B cell epitopes of ovalbumin protein The ovalbumin peptide 323-339 was obtained from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505; part number 4065609). Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702; product catalog number R1017). PLGA with a 3: 1 lactide: glycolide ratio and an intrinsic viscosity of 0.75 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211; product code 7525 DLG 7A). Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (Product No. 1.41350.1001).

  Solution 1: 20 mg / mL ovalbumin peptide 323-339 in dilute aqueous hydrochloric acid. A solution was prepared by dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution at room temperature. Solution 2: 50 mg / mL rapamycin in methylene chloride. A solution was prepared by dissolving rapamycin in pure methylene chloride. Solution 3: 100 mg / mL PLGA in methylene chloride. A solution was prepared by dissolving PLGA in pure methylene chloride. Solution 4: 50 mg / mL polyvinyl alcohol in 100 mM pH 8 phosphate buffer.

Method for Preparing Synthetic Nanocarriers Containing Rapamycin and Ovalbumin (323-339) A first water-in-oil emulsion was first prepared. Solution 1 (0.2 mL), Solution 2 (0.2 mL), and Solution 3 (1.0 mL) are combined in a small pressure tube using a Branson Digital Sonifier 250 for 40 seconds at 50% amplitude. W1 / O1 was prepared by decomposing with ultrasound. Next, Solution 4 (3.0 mL) is combined with the first W1 / O1 emulsion, vortexed for 10 seconds, and sonicated with a Branson Digital Sonifier 250 for 60 seconds at 30% amplitude. To prepare a second emulsion (W1 / O1 / W2).

  The W1 / O1 / W2 emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate the methylene chloride and form a synthetic nanocarrier. By transferring the synthetic nanocarrier suspension to a centrifuge tube, centrifuging at 21,000 × g and 4 ° C. for 1 hour, removing the supernatant, and resuspending the pellet in phosphate buffered saline, A portion of the synthetic nanocarrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline for a final synthetic nanocarrier dispersion of approximately 10 mg / mL.

  The amount of peptide and rapamycin in the synthetic nanocarrier was determined by HPLC analysis. The total mass of the dried synthetic nanocarrier per mL of suspension was measured by gravimetry.

Method for Producing Synthetic Nanocarriers Containing Rapamycin A first water-in-oil emulsion was first prepared. A 0.13M hydrochloric acid solution (0.2 mL), solution 2 (0.2 mL), and solution 3 (1.0 mL) were combined in a small pressure tube using a Branson Digital Sonifier 250 with 50% W1 / O1 was prepared by sonicating for 40 seconds in amplitude. Next, Solution 4 (3.0 mL) is combined with the first W1 / O1 emulsion, vortexed for 10 seconds, and sonicated with a Branson Digital Sonifier 250 for 60 seconds at 30% amplitude. To prepare a second emulsion (W1 / O1 / W2).

  The W1 / O1 / W2 emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate the methylene chloride and form a synthetic nanocarrier. By transferring the synthetic nanocarrier suspension to a centrifuge tube, centrifuging at 21,000 × g and 4 ° C. for 1 hour, removing the supernatant, and resuspending the pellet in phosphate buffered saline, A portion of the synthetic nanocarrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline for a final synthetic nanocarrier dispersion of approximately 10 mg / mL.

  The amount of rapamycin in the synthetic nanocarrier was determined by HPLC analysis. The total mass of the dried synthetic nanocarrier per mL of suspension was measured by gravimetry.

Method for Measuring Rapamycin Loading Approximately 3 mg of synthetic nanocarrier was collected and centrifuged to separate the supernatant from the synthetic nanocarrier pellet. Acetonitrile was added to the pellet and the sample was sonicated and centrifuged to remove any insoluble material. The supernatant and pellet were injected into RP-HPLC and the absorbance was read at 278 nm. The μg found in the pellet was used to calculate% uptake (load), and the supernatant and pellet μg were used to calculate the total recovered μg.

Method for Measuring Ovalbumin (323-339) Loading Approximately 3 mg of synthetic nanocarrier was collected and centrifuged to separate the supernatant from the synthetic nanocarrier pellet. Trifluoroethanol is added to the pellet, the sample is sonicated to dissolve the polymer, 0.2% trifluoroacetic acid is added, the sample is sonicated and then centrifuged to remove any Insoluble material was removed. The supernatant and pellet were injected into RP-HPLC and the absorbance was read at 215 nm. The μg found in the pellet was used to calculate% uptake (load), and the supernatant and pellet μg were used to calculate the total recovered μg.

Antigen-specific tolerogenic dendritic cell (tDC) activity on Treg cell development This assay has a transgenic T cell receptor specific for immunodominant ovalbumin peptide (323-339) The use of OTII mice was included. To generate antigen-specific tDC, CD11c ⁺ splenocytes were isolated and ovalbumin peptide (323-339) was added in vitro at 1 μg / mL or without antigen. Soluble or nanocarrier-encapsulated rapamycin was then added to the DC for 2 hours, which was then washed thoroughly to remove free rapamycin from the culture. Purified responder CD4 ⁺ CD25 ⁻ cells were isolated from OTII mice and added to tDC at a T: 1 to DC ratio of 10: 1. Next, a mixture of tDC and OTII T cells was cultured for 4-5 days, and the frequency of Treg cells (CD4 ⁺ CD25highFoxP3 ⁺ ) was analyzed by flow cytometry as shown in FIG. Regions were selected based on isotype controls.

Method for measuring the dimensions of the nanocarriers The dimensions of the synthetic nanocarriers were measured by dynamic light scattering (DLS). The suspension of synthetic nanocarrier was diluted with purified water to obtain a final synthetic nanocarrier suspension concentration of about 0.01-0.1 mg / mL. The diluted suspension was prepared directly in a suitable cuvette for DLS analysis. Next, the cuvettes were purchased from Brookhaven Instruments Corp. In ZetaPALS, equilibrated to 25 ° C. and then scanned for a sufficient amount of time to obtain a stable and reproducible distribution based on appropriate inputs for media viscosity and sample refractive index. Next, the effective diameter, or average of the distribution, was reported.

Results In the proof-of-concept experiment, the tolerance-inducing drug rapamycin was used in combination with the class II-conjugated ovalbumin peptide 323-339. Rapamycin is an immunosuppressive agent used to suppress allogeneic transplant rejection and an inhibitor of mTOR, which regulates several cellular functions including APC and T cell behavior It is an agent. Synthetic nanocarriers were prepared according to the above. The representative examples are described in detail in the following tables (Tables 2 to 4).

The results from a representative flow cytometry analysis show an increase in the number of CD4 ⁺ CD25 highFoxP3 ⁺ cells when DCs are treated with free rapamycin and free ovalbumin (323-339) (FIG. 1).

Free rapamycin or synthetic nanocarriers containing rapamycin were combined with free soluble ovalbumin (323-339) to evaluate the induction of tDC (FIG. 2). It was found that nanocarriers containing rapamycin combined with free ovalbumin (323-339) induce Treg generation. Briefly, dendritic cells (CD11c ⁺ splenocytes) were isolated and separated from ovalbumin (323-339) peptide and soluble or nanocarrier-encapsulated rapamycin (synthetic nanocarrier numbers 13, 14, 15 and 16). After culturing in combination for 2 hours, antigen-specific tDC was obtained by washing thoroughly. Purified responder CD4 ⁺ CD25 ⁻ cells were isolated from OTII mice and added to tDC. Next, the mixture of tDC and OTII T cells was cultured for 4-5 days, and the frequency of Treg cells (CD4 ⁺ CD25 highFoxP3 ⁺ ) was analyzed by flow cytometry. The data show a dose-dependent increase in CD4 ⁺ CD25highFoxP3 ⁺ for both free rapamycin and nanocarrier-encapsulated rapamycin, suggesting induction of Treg by DCs treated with rapamycin nanocarrier.

Various nanocarrier compositions were used to evaluate the induction of tDC (FIG. 3) and to demonstrate the induction of Treg. Nanocarriers with co-encapsulated rapamycin and ovalbumin (323-339) peptide induced higher induction of FoxP3-expressing cells than either unstimulated (1.3%) or rapamycin alone (2.7%) (6. 5%). Interestingly, two separate nanocarrier compositions containing synthetic rapamycin alone (synthetic nanocarrier numbers 7 and 8) were mixed with free ovalbumin (323-339) peptide (12.7% and 17.7 respectively). Better induction of FoxP3-expressing cells (22.4% and 27.2%, respectively) when combined with a population of synthetic nanocarriers containing ovalbumin (323-339) Indicated. Overall, the data show an increase in CD4 ⁺ CD25highFoxP3 ⁺ when using nanocarrier-encapsulated rapamycin, with a better response indicating either co-encapsulated ovalbumin (323-339) peptide or mixed ovalbumin (323 -339) found with nanocarriers containing any of the peptides.

Example 6: Evaluation of tolerogenic immune response by T cell phenotype analysis (example of theoretical experiment)
The composition of the present invention is dissolved in phosphate buffered saline (PBS) and injected intramuscularly into female Lewis rats in 0.1-0.2 ml containing 500 μg of the composition. A control group of rats receives 0.1-0.2 ml PBS. Nine to ten days after injection, spleens and lymph nodes are harvested from rats and single cell suspensions are obtained by maceration through a 40 μm nylon cell strainer. Stain samples in PBS (1% FCS) with appropriate dilution of relevant monoclonal antibodies. Propidium iodide stained cells are excluded from the analysis. Samples are acquired with an LSR2 flow cytometer (BD Biosciences, USA) and analyzed using FACS Diva software. The expression of markers CD4, CD25high and FoxP3 is analyzed in the cells. The presence of CD4 ⁺ CD25highFoxP3 ⁺ cells suggests the induction of CD4 + Treg cells.

Example 7: Evaluation of tolerogenic immune response to APC-presentable antigen in vivo (example of theoretical experiment)
Balb / c mice are immunized with an APC-presentable antigen in incomplete Freund's adjuvant to induce T cell proliferation (eg, CD4 ⁺ T cells) and evaluate its level. Thereafter, the composition of the present invention comprising an APC-presentable antigen and an immunosuppressive agent is administered subcutaneously in a dose-dependent manner. The same mouse is then re-exposed to the APC presentable antigen and the level of T cell proliferation is again assessed. Next, changes in the T cell population are monitored by a decrease in T cell proliferation upon subsequent loading with an APC presentable antigen that induces a tolerogenic immune response.

Example 8 Evaluation of Tolerogenic Immune Response with Synthetic Nanocarriers Containing Immunosuppressant and APC Presentable Antigen in vivo Materials and Methods for Synthetic Nanocarrier Production Nanocarrier 1
Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702; product catalog number R1017). PLGA with a 3: 1 lactide: glycolide ratio and an intrinsic viscosity of 0.75 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211; product code 7525 DLG 7A). A PLA-PEG block copolymer of about 5,000 Da PEG block and about 20,000 Da PLA block was synthesized. Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (Product No. 1.41350.1001).

The solution was prepared as follows:
Solution 1: 50 mg / mL rapamycin in methylene chloride. A solution was prepared by dissolving rapamycin in pure methylene chloride. Solution 2: 100 mg / mL PLGA in methylene chloride. A solution was prepared by dissolving PLGA in pure methylene chloride. Solution 3: 100 mg / mL PLA-PEG in methylene chloride. A solution was prepared by dissolving PLA-PEG in pure methylene chloride. Solution 4: 50 mg / mL polyvinyl alcohol in 100 mM pH 8 phosphate buffer.

  Nanocarriers were prepared using an oil-in-water emulsion. Solution 1 (0.2 mL), Solution 2 (0.75 mL), Solution 3 (0.25 mL), and Solution 4 (3 mL) were combined in a small pressure tube using a Branson Digital Sonifier 250, 30 An O / W emulsion was prepared by sonication for 60 seconds at% amplitude. The O / W emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate the methylene chloride and form nanocarriers. The nanocarrier suspension is transferred to a centrifuge tube, centrifuged at 21,000 × g and 4 ° C. for 45 minutes, the supernatant is removed, and the pellet is resuspended in phosphate buffered saline. A part of the nanocarrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline for a final nanocarrier dispersion of approximately 10 mg / mL.

  The nanocarrier size was measured by dynamic light scattering. The amount of rapamycin in the nanocarrier was measured by HPLC analysis. The total mass of dry nanocarrier per mL of suspension was measured by gravimetric method.

Nanocarrier 2
Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and B cell epitope of ovalbumin protein, was obtained from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505; part number 4065609). PLGA with a 3: 1 lactide: glycolide ratio and an intrinsic viscosity of 0.75 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211; product code 7525 DLG 7A). A PLA-PEG block copolymer of about 5,000 Da PEG block and about 20,000 Da PLA block was synthesized. Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (Product No. 1.41350.1001).

The solution was prepared as follows:
Solution 1: 20 mg / mL ovalbumin peptide 323-339 in dilute aqueous hydrochloric acid. A solution was prepared by dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution at room temperature. Solution 2: 100 mg / mL PLGA in methylene chloride. A solution was prepared by dissolving PLGA in pure methylene chloride. Solution 3: 100 mg / mL PLA-PEG in methylene chloride. A solution was prepared by dissolving PLA-PEG in pure methylene chloride. Solution 4: 50 mg / mL polyvinyl alcohol in 100 mM pH 8 phosphate buffer.

  A first water-in-oil emulsion was first prepared. Solution 1 (0.2 mL), Solution 2 (0.75 mL), and Solution 3 (0.25 mL) are combined in a small pressure tube using a Branson Digital Sonifier 250 at 50% amplitude for 40 seconds. W1 / O1 was prepared by decomposing with ultrasound. Next, Solution 4 (3.0 mL) is combined with the first W1 / O1 emulsion, vortexed for 10 seconds, and sonicated with a Branson Digital Sonifier 250 for 60 seconds at 30% amplitude. To prepare a second emulsion (W1 / O1 / W2).

  The W1 / O1 / W2 emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate the methylene chloride and form nanocarriers. Transfer the nanocarrier suspension to a centrifuge tube, centrifuge at 75,600 × g and 4 ° C. for 35 minutes, remove the supernatant, and resuspend the pellet in phosphate buffered saline. A part of the carrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline for a final nanocarrier dispersion of approximately 10 mg / mL.

  The nanocarrier size was measured by dynamic light scattering. The amount of peptide in the nanocarrier was measured by HPLC analysis. The total mass of dry nanocarrier per mL of suspension was measured by gravimetric method.

Nanocarrier 3
Simvastatin was obtained from LKT Laboratories, Inc. (2233 University Avenue West, St. Paul, MN 55114; product catalog number S3449). PLGA with a 3: 1 lactide: glycolide ratio and an intrinsic viscosity of 0.75 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211; product code 7525 DLG 7A). A PLA-PEG block copolymer of about 5,000 Da PEG block and about 20,000 Da PLA block was synthesized. Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (Product No. 1.41350.1001).

The solution was prepared as follows:
Solution 1: 50 mg / mL simvastatin in methylene chloride. A solution was prepared by dissolving simvastatin in pure methylene chloride. Solution 2: 100 mg / mL PLGA in methylene chloride. A solution was prepared by dissolving PLGA in pure methylene chloride. Solution 3: 100 mg / mL PLA-PEG in methylene chloride. A solution was prepared by dissolving PLA-PEG in pure methylene chloride. Solution 4: 50 mg / mL polyvinyl alcohol in 100 mM pH 8 phosphate buffer.

  Nanocarriers were prepared using an oil-in-water emulsion. Solution 1 (0.15 mL), Solution 2 (0.75 mL), Solution 3 (0.25 mL), and Solution 4 (3 mL) are combined in a small pressure tube using a Branson Digital Sonifier 250. An O / W emulsion was prepared by sonication for 60 seconds at% amplitude. The O / W emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate the methylene chloride and form nanocarriers. Transfer the nanocarrier suspension to a centrifuge tube, centrifuge at 75,600 × g and 4 ° C. for 35 minutes, remove the supernatant, and resuspend the pellet in phosphate buffered saline. A part of the carrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline for a final nanocarrier dispersion of approximately 10 mg / mL.

  The nanocarrier size was measured by dynamic light scattering. The amount of simvastatin in the nanocarrier was measured by HPLC analysis. The total mass of dry nanocarrier per mL of suspension was measured by gravimetric method.

Nanocarrier 4
Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and B cell epitope of ovalbumin protein, was obtained from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505; part number 4065609). Rapamycin was purchased from TSZ CHEM (185 Wilson Street, Framingham, MA 01702; product catalog number R1017). PLGA with a 3: 1 lactide: glycolide ratio and an intrinsic viscosity of 0.75 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211; product code 7525 DLG 7A). A PLA-PEG block copolymer of about 5,000 Da PEG block and about 20,000 Da PLA block was synthesized. Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (Product No. 1.41350.1001).

The solution was prepared as follows:
Solution 1: 20 mg / mL ovalbumin peptide 323-339 in dilute aqueous hydrochloric acid. A solution was prepared by dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution at room temperature. Solution 2: 50 mg / mL rapamycin in methylene chloride. A solution was prepared by dissolving rapamycin in pure methylene chloride. Solution 3: 100 mg / mL PLGA in methylene chloride. A solution was prepared by dissolving PLGA in pure methylene chloride. Solution 4: 100 mg / mL PLA-PEG in methylene chloride. A solution was prepared by dissolving PLA-PEG in pure methylene chloride. Solution 5: 50 mg / mL polyvinyl alcohol in 100 mM pH 8 phosphate buffer.

  A first water-in-oil emulsion was first prepared. Solution 1 (0.2 mL), Solution 2 (0.2 mL), Solution 3 (0.75 mL), and Solution 4 (0.25 mL) are combined in a small pressure tube using a Branson Digital Sonifier 250. W1 / O1 was prepared by sonication at 50% amplitude for 40 seconds. Next, Solution 5 (3.0 mL) is combined with the first W1 / O1 emulsion, vortexed for 10 seconds, and sonicated with a Branson Digital Sonifier 250 for 60 seconds at 30% amplitude. To prepare a second emulsion (W1 / O1 / W2).

  The W1 / O1 / W2 emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate the methylene chloride and form nanocarriers. The nanocarrier suspension is transferred to a centrifuge tube, centrifuged at 21,000 × g and 4 ° C. for 45 minutes, the supernatant is removed, and the pellet is resuspended in phosphate buffered saline. A part of the carrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline for a final nanocarrier dispersion of approximately 10 mg / mL.

  The nanocarrier size was measured by dynamic light scattering. The amount of peptide and rapamycin in the nanocarrier was determined by HPLC analysis. The total mass of dry nanocarrier per mL of suspension was measured by gravimetric method.

Nanocarrier 5
Ovalbumin peptide 323-339, a 17 amino acid peptide known to be a T and B cell epitope of ovalbumin protein, was obtained from Bachem Americas Inc. (3132 Kashiwa Street, Torrance CA 90505; part number 4065609). Simvastatin was obtained from LKT Laboratories, Inc. (2233 University Avenue West, St. Paul, MN 55114; product catalog number S3449). PLGA with a 3: 1 lactide: glycolide ratio and an intrinsic viscosity of 0.75 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211; product code 7525 DLG 7A). A PLA-PEG block copolymer of about 5,000 Da PEG block and about 20,000 Da PLA block was synthesized. Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (Product No. 1.41350.1001).

The solution was prepared as follows:
Solution 1: 20 mg / mL ovalbumin peptide 323-339 in dilute aqueous hydrochloric acid. A solution was prepared by dissolving ovalbumin peptide in 0.13 M hydrochloric acid solution at room temperature. Solution 2: 50 mg / mL simvastatin in methylene chloride. A solution was prepared by dissolving simvastatin in pure methylene chloride. Solution 3: 100 mg / mL PLGA in methylene chloride. A solution was prepared by dissolving PLGA in pure methylene chloride. Solution 4: 100 mg / mL PLA-PEG in methylene chloride. A solution was prepared by dissolving PLA-PEG in pure methylene chloride. Solution 5: 50 mg / mL polyvinyl alcohol in 100 mM pH 8 phosphate buffer.

  A first water-in-oil emulsion was first prepared. Solution 1 (0.2 mL), Solution 2 (0.15 mL), Solution 3 (0.75 mL), and Solution 4 (0.25 mL) are combined in a small pressure tube using a Branson Digital Sonifier 250. W1 / O1 was prepared by sonication at 50% amplitude for 40 seconds. Next, Solution 5 (3.0 mL) is combined with the first W1 / O1 emulsion, vortexed for 10 seconds, and sonicated with a Branson Digital Sonifier 250 for 60 seconds at 30% amplitude. To prepare a second emulsion (W1 / O1 / W2).

  The W1 / O1 / W2 emulsion was added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate the methylene chloride and form nanocarriers. Transfer the nanocarrier suspension to a centrifuge tube, centrifuge at 75,600 × g and 4 ° C. for 35 minutes, remove the supernatant, and resuspend the pellet in phosphate buffered saline. A part of the carrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline for a final nanocarrier dispersion of approximately 10 mg / mL.

  The nanocarrier size was measured by dynamic light scattering. The amount of peptide and simvastatin in the nanocarrier was determined by HPLC analysis. The total mass of dry nanocarrier per mL of suspension was measured by gravimetric method.

In vivo administration 1
B6. Spleens from Cg-Tg (TcraTcrb) 425 Cbn / J (OTII) and C57BL / 6 (B6) mice are collected, mechanically dissociated, and filtered separately through a 70 μM sieve to obtain a single cell suspension. It was. The purified CD4 ⁺ CD25 − cells were then extracted in a two-step process. Spleen cells were first labeled with CD4 ⁺ T cell isolation kit II using a Miltenyi Biotec AutoMACS magnetic cell sorter and CD25 ⁺ cells were removed from unlabeled fractions using a CD25 removal kit. . Purified B6 cells were stained with the cell dye, carboxyfluorescein succinimidyl ester (CFSE), and then mixed at a concentration equal to purified OTII cells. Next, they are designated as B6. SJL-Ptprc ^{a /} BoyAi (CD45.1) recipient mice were injected intravenously.

The next day, recipient CD45.1 mice were treated with targeted tolerogenic synthetic vaccine particles (t ² SVP). They were loaded with the combination of ovalbumin peptide (323-339) (OVA ^323-339 ), rapamycin (Rapa) and / or simvastatin (Simva) and administered subcutaneously.

  This injection was a tolerogenic treatment, followed by 4 more injections, each 2 weeks apart. After the treatment schedule is complete, the recipient CD45.1 animals are sacrificed and their spleen and popliteal lymph nodes are harvested, mechanically dissociated, filtered separately through a 70 μM sieve, and single cell suspensions are obtained. A turbid liquid was obtained. Red blood cells (RBC) were removed from spleen cells by incubation with RBC lysis buffer (Stem Cell Technologies) and cell counts were performed in both spleen and lymph nodes.

Spleen or lymph node cells are cultured in CM (complete medium) supplemented with 10 U / ml IL-2 and 0.3 × 10 ⁶ cells per well in 96 well round bottom (RB) plates. Re-stimulated with OPII and incubated at 37 ° C., 5% CO ₂ . Cells were split on day 2 and harvested on day 5. Supernatants were collected and frozen while cells were stained for phenotypic analysis by flow cytometry. Cells were analyzed on a Becton Dickinson FacsCanto flow cytometer.

In vivo administration 2
B6. Spleens from Cg-Tg (TcraTcrb) 425 Cbn / J (OTII) and C57BL / 6 (B6) mice are collected, mechanically dissociated, and filtered separately through a 70 μM sieve to obtain a single cell suspension. It was. The purified CD4 ⁺ CD25 − cells were then extracted in a two-step process using a Miltenyi Biotec AutoMACS magnetic cell sorter. Spleen cells were labeled using Miltenyi's CD4 ⁺ T cell isolation kit II. Next, CD25 ⁺ cells were removed from the unlabeled CD4 + T cell fraction using a CD25 removal kit. Next, purified CD4 cells from B6 mice were stained with the cell dye, carboxyfluorescein succinimidyl ester (CFSE), and then mixed at a concentration equal to purified OTII cells. Next, they are designated as B6. SJL-Ptprc ^{a /} BoyAi (CD45.1) recipient mice were injected intravenously.

The next day, recipient CD45.1 mice were treated with targeted tolerogenic synthetic vaccine particles. They included a combination of ovalbumin peptide (323-339) (OVA ^323-339 ), rapamycin (Rapa) and simvastatin (Simva) and were administered subcutaneously or intravenously.

  After the treatment schedule is complete, the recipient CD45.1 animals are sacrificed and their spleen and popliteal lymph nodes are harvested, mechanically dissociated, filtered separately through a 70 μM sieve, and single cell suspensions are obtained. A turbid liquid was obtained. Red blood cells (RBC) were removed from spleen cells by incorporation with RBC lysis buffer (Stem Cell Technologies) and cell counts were performed in both spleen and lymph nodes.

Spleen or lymph node cells are cultured in CM supplemented with 10 U / ml IL-2 and 1 x M OPII at 0.3 x 10 ⁶ cells per well in a 96 well round bottom (RB) plate. And incubated again at 37 ° C., 5% CO ₂ . Cells were split on day 2 and harvested on day 5. Supernatants were collected and frozen while cells were stained for phenotypic analysis by flow cytometry. Cells were analyzed on a Becton Dickinson FacsCanto flow cytometer.

Results The results are shown in FIGS. 4 and 5 (immunomodulator 1: rapamycin; immunomodulator 2: simvastatin). These figures show in vivo effects and show that the number of immune cells is reduced by synthetic nanocarriers containing antigens and immunosuppressive agents when compared to antigen-only nanocarriers. FIG. 5 shows an increase in the percentage of immune cells expressing FoxP3 by synthetic nanocarriers containing antigen and immunosuppressive agent.

Example 9: Evaluation of CD69 activation by synthetic nanocarriers containing immunosuppressive agents Nanocarriers α-galactosylceramide (KRN7000) was obtained from Avanti Polar Lipids, Inc. (700 Industrial Park Drive Alabaster, Alabama 35007-9105; catalog number 867000P). PLGA having a 1: 1 lactide: glycolide ratio and an intrinsic viscosity of 0.45 dL / g was purchased from SurModics Pharmaceuticals (756 Tom Martin Drive, Birmingham, AL 35211; product code 5050 DLG 4.5A). Polyvinyl alcohol (85-89% hydrolyzed) was purchased from EMD Chemicals (Product No. 1.41350.1001).

The solution was prepared as follows:
Solution 1: 2 mg / mL KRN7000 in dimethyl sulfoxide (DMSO). A solution was prepared by dissolving the dried lipid in pure DMSO. Solution 2: 100 mg / mL PLGA in methylene chloride. A solution was prepared by dissolving PLGA in pure methylene chloride. Solution 3: 50 mg / mL polyvinyl alcohol in 100 mM pH 8 phosphate buffer.

  Solution 1 (1 mL), Solution 2 (1 mL), Solution 3 (3 mL) are combined in a small pressure tube immersed in an ice-water bath using a Branson Digital Sonifier model 250 for 60 seconds at 30% amplitude. A water-in-oil emulsion (W / O) was prepared by ultrasonic decomposition. The W / O emulsion was then added to a beaker containing 70 mM pH 8 phosphate buffer (30 mL) and stirred at room temperature for 2 hours to evaporate the methylene chloride and form nanocarriers. Transfer the nanocarrier suspension to a centrifuge tube, centrifuge at 75,600 × g for 45 minutes at 4 ° C., remove the supernatant, and resuspend the pellet in phosphate buffered saline. A part of the nanocarrier was washed. The washing procedure was repeated and the pellet was resuspended in phosphate buffered saline for a final nanocarrier dispersion of approximately 10 mg / mL. The nanocarrier size was measured by dynamic light scattering. The amount of KRN7000 in the nanocarrier is reported as the theoretical loading when there is no loss during processing. The total mass of dry nanocarrier per mL of suspension (NP concentrate) was measured by gravimetric method.

Immunization Mice were assigned to groups receiving PBS alone, sol aGC + solRAPA, sol aGC + NP RAPA. Mice were injected at 9 am and Brefeldin A was administered intravenously at 9.30 AM to prevent the release of produced intracellular cytokines. At 1 pm, mice were sacrificed and the liver was perfused with PBS, which was processed to obtain a single cell suspension enriched in lymphocytes. Cells were stained with the cell surface marker for iNKT cells (agd loaded CD1d tetramer) and the T cell receptor (TCRb) and the activation marker CD69. Cells were acquired on a FacsCanto flow cytometer and analyzed by FlowJo.

Results iNKT cells from mice administered aGC were activated as evidenced by cytokine production on their surface and upregulation of CD69. Both soluble and nanocarrier rapamycin significantly reduced the surface level of CD69 in iNKT cells. This suggests that the cells were less activated after treatment compared to PBS treated controls. The result is shown in FIG.

Example 10: Mesoporous Silica-Gold Core-Shell Nanocarrier Containing Ovalbumin (Theoretical Experimental Example)
A mesoporous SiO2 nanoparticle core is produced by a sol-gel method. Hexadecyltrimethylammonium bromide (CTAB) (0.5 g) is dissolved in deionized water (500 mL) and then 2 M aqueous NaOH (3.5 mL) is added to the CTAB solution. The solution is stirred for 30 minutes and then tetraethoxysilane (TEOS) (2.5 mL) is added to the solution. The resulting gel is stirred at a temperature of 80 ° C. for 3 hours. The resulting white precipitate is captured by filtration, then washed with deionized water and dried at room temperature. The remaining surfactant is then extracted from the particles by suspending in HCl in ethanol overnight. The particles are washed with ethanol, centrifuged and redispersed by sonication. Repeat this washing procedure two more times.

  Next, the SiO2 nanoparticles are functionalized with amino groups using (3-aminopropyl) -triethoxysilane (APTMS). To do this, the particles are suspended in ethanol (30 mL) and APTMS (50 μL) is added to the suspension. The suspension is left at room temperature for 2 hours, then boiled for 4 hours and the volume is kept constant by adding ethanol periodically. Remaining reactants are removed by five cycles of washing by centrifugation and redispersion in pure ethanol.

  In the separation reaction, a gold seed having a diameter of 1 to 4 nm is generated. All water used in this reaction is first deionized and then distilled from the glass. Add water (45.5 mL) to a 100 mL round bottom flask. While stirring, a 0.2 M aqueous NaOH solution (1.5 mL) is added, followed by a 1% aqueous solution (1.0 mL) of tetrakis (hydroxymethyl) phosphonium chloride (THPC). Two minutes after the addition of the THPC solution, a 10 mg / mL aqueous solution (2 mL) of chloroauric acid aged for at least 15 minutes is added. The gold seed is purified by dialysis against water.

  To form the core-shell nanocarrier, the amino-functionalized SiO2 nanoparticles formed above are first mixed with a gold seed at room temperature for 2 hours. The gold-decorated SiO2 particles are collected by centrifugation and mixed with an aqueous solution of chloroauric acid and potassium bicarbonate to form a gold shell. The particles are then washed by centrifugation and redispersed in water. Thiolated ovalbumin (made by treating ovalbumin with 2-iminothiolane hydrochloride) is loaded by suspending the particles in a solution of thiolated ovalbumin (1 mg / L) for 72 hours. The particles are then pellet washed with 1 × PBS (pH 7.4) to remove free protein. The resulting silica-gold core-shell nanocarrier containing ovalbumin is then resuspended in 1 × PBS for further analysis and assay.

Example 11: Liposomes containing rapamycin and ovalbumin (theoretical experimental example)
Liposomes are formed by thin film hydration. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (32 μmol), cholesterol (32 μmol), and rapamycin (6.4 μmol) are dissolved in pure chloroform (3 mL). This lipid solution is added to a 10 mL glass tube and the solvent is removed under a stream of nitrogen gas and dried under vacuum for 6 hours. Multilamellar vesicles are obtained by hydration of thin films with 2.0 ml of 25 mM MOPS buffer (pH 8.5) containing excess ovalbumin. Vortex the tube until the lipid film peels off the tube surface. Ten cycles of freezing (liquid nitrogen) and thawing (30 ° C. water bath) are performed to break down the multilamellar vesicles into monolayer membranes. The sample is then diluted to 1 ml in 25 mM MOPS buffer (pH 8.5). By passing the sample 10 times through a 200 nm pore polycarbonate filter, the size of the resulting liposomes is made uniform by extrusion. The resulting liposomes are then used for further analysis and bioassay.

Example 12: Polymer nanocarrier composed of polyamino acids modified with surface-conjugated ovalbumin (theoretical experimental example)
Step 1. Preparation of poly (γ-glutamic acid) (γ-PGA) modified with L-phenylalanine ethyl ester (L-PAE): 4.7 mmol units of γ-PGA (Mn = 300 kD) was added to 0.3N NaHCO 3 aqueous solution ( 50 mL). L-PAE (4.7 mmol) and EDC. HCl (4.7 mmol) is added to the solution and stirred at 4C for 30 minutes. The solution is then maintained at room temperature with stirring for 24 hours. Low molecular weight chemicals are removed by dialysis using a dialysis membrane with a MWCO of 50 kD. The obtained γ-PGA-graft-L-PAE is obtained by lyophilization.

  Step 2. Preparation of nanoparticles from γ-PGA-graft-L-PAE polymer: Nanoparticles composed of γ-PGA-graft-L-PAE are prepared by precipitation and dialysis methods. γ-PGA-graft-L-PAE (20 mg) was dissolved in 2 ml of DMSO and then 2 ml of water was added to form a translucent solution. The solution is then dialyzed against distilled water using a cellulose membrane tube (50,000 MWCO) to form nanoparticles and the organic solvent is removed for 72 hours at room temperature. Distilled water is changed at 12 hour intervals. The resulting nanoparticle solution (10 mg / mL in water) is then used for antigen conjugation.

  Step 3. Ovalbumin conjugation to γ-PGA nanoparticles: The surface carboxylic acid groups of γ-PGA nanoparticles (10 mg / ml) were allowed to react for 2 hours at ambient temperature with EDC and NHS (10 mg / mL in phosphate buffer, pH 5. First, it is activated by 8). After pellet washing to remove excess EDC / NHS, the activated nanoparticles were mixed with 1 mL ovalbumin (10 mg / ml) in phosphate buffered saline (PBS, pH 7.4). Incubate the mixture at 4-8C for 24 hours. The resulting ovalbumin conjugated γ-PGA nanoparticles are washed twice with PBS and resuspended at 5 mg / mL in PBS for further analysis and bioassay.

Example 13: Erythropoietin (EPO) -encapsulated γ-PGA nanoparticles (theoretical experiment example)
To prepare EPO encapsulated γ-PGA nanoparticles, 0.25-4 mg of EPO was dissolved in 1 mL of PBS (pH 7.4) and 1 mL of γ-PGA-graft-L-PAE (10 mg / mL in DMSO). mL) is added to the EPO solution. The resulting solution is centrifuged at 14,000 × g for 15 minutes and rinsed repeatedly with PBS. The resulting EPO encapsulated γ-PGA nanoparticles are then resuspended in PBS (5 mg / mL) for further analysis and bioassay.

Example 14: Preparation of gold nanocarrier (AuNC) containing ovalbumin (example of theoretical experiment)
Step 1. Formation of gold NC (AuNC): 500 mL of an aqueous solution of 1 mM HAuCl4 is heated to reflux for 10 minutes with vigorous stirring in a 1 L round bottom flask equipped with a condenser. Next, 50 mL of 40 mM trisodium citrate solution is quickly added to the stirring solution. The resulting dark wine red solution is kept at reflux for 25-30 minutes, the heat is removed and the solution is cooled to room temperature. The solution is then filtered through a 0.8 μm membrane filter to obtain an AuNC solution. AuNC is characterized using visible spectroscopy and transmission electron microscopy. AuNC is about 20 nm in diameter, capped with citrate and has a peak absorption at 520 nm.

  Step 2. Conjugation of ovalbumin to AuNC: a solution of 150 μl of thiolated ovalbumin (10 μ 中 in 10 mM pH 9.0 carbonate buffer) gold nanocarrier (1.16 nM) capped with 1 mL of 20 nm diameter citrate ) To produce a thiol to gold molar ratio of 2500: 1. The mixture is stirred at room temperature for 1 hour under argon to allow complete exchange of thiol and citrate on the gold nanocarrier. The ovalbumin and AuNC on the surface are then purified by centrifugation at 12,000 g for 30 minutes. The supernatant is decanted and the pellet containing AuNC-ovalbumin is then pellet washed with 1 × PBS buffer. The purified gold-ovalbumin nanocarrier is then resuspended in a suitable buffer for further analysis and bioassay.

F. References Listing of any reference herein not an admission that the reference or it teachings is / are prior art.
Differential impact of mammarian target of rapamycin inhibition on CD4 + CD25 + Foxp3 + regulatory T cells compared with conjugation cell CD4 +. Zeiser R, Leveson-Gower DB, Zambricki EA, Kambham N, Beilhack A, Loh J, Hou JZ, Negrin RS. Blood. 2008 Jan 1; 111 (1): 453-62.
Combined administration of a mutant TGF-beta1 / Fc and rapamycin promoters induc- tion of regulatory T cells and islet alligator tolerance. Zhang W, Zhang D, Shen M, Liu Y, Tian Y, Thomson AW, Lee WP, Zheng XX. J Immunol. 2010 Oct 15; 185 (8): 4750-9.
Delivery of rapamicin by PLGA nanoparticulates enhances supplements on dendritic cells. Haddadi A, Elamanchili P, Lavasanifar A, Das S, Shapiro J, Samuel J. et al. J Biomed Mater Res A.M. 2008 Mar 15; 84 (4): 885-98.
Delivery of rapamycin-loaded nanoparticulate down regulates ICAM-1 expression and maintenance an immunosuppressive in human pump. CD34 + Das S, Haddadi A, Venice S, Samuel J. et al. J Biomed Mater Res A.M. 2008 Jun 15; 85 (4): 983-92.
Mammalian target of rapamycin inhibition and alloantigen-specific regulatory T cells synergize to promote long-term graft recirculinp Raimondi G, Sumpter TL, Matta BM, Pillai M, Corbitt N, Vodovotz Y, Wang Z, Thomson AW. J Immunol. 2010 Jan 15; 184 (2): 624-36.
mTOR and GSK-3 shape the CD4 + T-cell stability and differentiation capacity of myloid DCs after exposure to LPS. Turnquist HR, Cardinal J, Macedo C, Rosborough BR, Sumpter TL, Geller DA, Metes D, Thomson AW. Blood. 2010 Jun 10; 115 (23): 4758-69.
Immunoregulatory functions of mTOR inhibition. Thomson AW, Turnquist HR, Raimondi G. Nat Rev Immunol. 2009 May; 9 (5): 324-37.
Rapamycin-conditioned donor dendritic cells differentiated CD4CD25 Foxp3 T cells in vitro with TGF-beta1 for islet translation. Pothoven KL, Kheradmand T, Yang Q, Houlihan JL, Zhang H, Degutes M, Miller SD, Luo X. Am J Transplant. 2010 Aug; 10 (8): 1774-84.
Yaguee, C.I. Aruebo M .; Santamaria, J .; NIR-enhanced drug release from porous Au / SiO ₂ nanoparticles. Chem. Commun. 46, 7513-7515 (2010).
Zeng, W.M. Qian, X .; Zhang, Y .; Yin, J .; Zhu, Z .; Organic modified mesoporous MCM-41 through solvary thermal process as drug delivery system. Mater. Res. Bull. 40, 766-772 (2005).
Oldenburg, S.M. J. et al. Westcott, S .; L. Averitt, R .; D. Halas, N .; J. et al. Surface enhanced Raman scattering in the near-infrared using metal nanoshell substrates. J. et al. Chem. Phys. 111, 4729-4735 (1999).
Duff, D.D. G. Baiker, A .; A new hydrosol of gold clusters. 1. Formation and particle size variation. Langmuir 9, 2301-2309 (1993).
Gregoriadis, G.M. McCorack B .; Obrenovic, M .; Saffie, R .; Brahim, Z .; Perrie, Y .; Vaccine entrapment in liposomes. Methods 19, 156-162 (1999).
Arulsudar, N .; Subramanian, N .; Mishra, P .; Sharma, R .; K. Murthy, R .; S. R. Preparation, characterization and biodistribution of ^99m Tc-labeled liposome encapsulated cyclosporine. J. et al. Drug Target. 11, 187-196 (2003).

Claims (44)

(I) a first population of synthetic nanocarriers coupled to an immunosuppressant;
(Ii) A composition comprising an APC-presentable antigen.   2. The composition of claim 1, wherein the immunosuppressive agent in the composition is an amount effective to produce a tolerogenic immune response against the APC presentable antigen.   3. The composition of claim 1 or 2, wherein the APC presentable antigen is bound to a synthetic nanocarrier of the first population of synthetic nanocarriers.   3. The composition of claim 1 or 2, wherein the APC presentable antigen is bound to a second population of synthetic nanocarriers of synthetic nanocarriers.   The average particle size distribution obtained using dynamic light scattering of the synthetic nanocarriers of the first and / or second population is a diameter of more than 100 nm. Composition.   6. The composition of claim 5, wherein the diameter is greater than 150 nm.   The composition of claim 6, wherein the diameter is greater than 200 nm.   The composition of claim 7, wherein the diameter is greater than 250 nm.   The composition of claim 8, wherein the diameter is greater than 300 nm.   The immunosuppressive agent is a statin, mTOR inhibitor, TGF-β signaling agent, corticosteroid, inhibitor of mitochondrial function, P38 inhibitor, NF-κβ inhibitor, adenosine receptor agonist, prostaglandin E2 agonist, The composition according to any one of claims 1 to 9, comprising a phosphodiesterase 4 inhibitor, an HDAC inhibitor or a proteasome inhibitor.   11. The composition of claim 10, wherein the mTOR inhibitor is rapamycin.   The composition according to any one of claims 1 to 11, wherein the APC-presentable antigen comprises an MHC class I-restricted and / or MHC class II-restricted epitope.   The composition according to any one of claims 1 to 11, wherein the APC-presentable antigen is a lipid that binds to CD1d.   The APC presentable antigen is a therapeutic protein or portion thereof, a self antigen or an allergen, or is associated with an autoimmune disease, inflammatory disease, allergy, organ or tissue rejection or graft-versus-host disease. The composition as described in any one of 1-13.   15. The immunosuppressant loading is 0.0001% to 50% (weight / weight) on average over the entire population of first synthetic nanocarriers. Composition.   16. The composition of claim 15, wherein the immunosuppressant loading is 0.1% to 10% (weight / weight) on average for the entire population of first synthetic nanocarriers.   17. The load of the APC presentable antigen is 0.0001% to 50% (weight / weight) on average over the entire population of first and / or second synthetic nanocarriers. The composition according to any one of the above.   18. The APC presentable antigen load is 0.1% to 10% (weight / weight) on average over the entire population of first and / or second synthetic nanocarriers. Composition.   The synthetic nanocarriers of the first and / or second population of synthetic nanocarriers are lipid nanoparticles, polymer nanoparticles, metal nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles Or the composition as described in any one of Claims 1-18 containing a peptide or protein particle | grains.   20. The polymer nanoparticle comprises a polymer that is a non-methoxy terminated pluronic polymer when the synthetic nanocarrier of the first and / or second population of synthetic nanocarriers comprises polymer nanoparticles. Composition.   21. A composition according to claim 19 or 20, wherein the polymer nanoparticles comprise polyester, polyester bonded to polyether, polyamino acid, polycarbonate, polyacetal, polyketal, polysaccharide, polyethyloxazoline or polyethyleneimine.   The composition according to claim 21, wherein the polyester comprises poly (lactic acid), poly (glycolic acid), poly (lactic acid-co-glycolic acid) or polycaprolactone.   23. A composition according to claim 21 or 22, wherein the polymer nanoparticles comprise polyester bonded to polyester and polyether.   24. The composition according to any one of claims 21 to 23, wherein the polyether comprises polyethylene glycol or polypropylene glycol.   The aspect ratios of the synthetic nanocarriers of the first and / or second population of synthetic nanocarriers are 1: 1, 1: 1.2, 1: 1.5, 1: 2, 1: 3, 1: 25. A composition according to any one of claims 1 to 24, greater than 5, 1: 7 or 1:10.   26. The composition according to any one of claims 1-25, further comprising a pharmaceutically acceptable excipient.   A dosage form comprising the composition according to any one of claims 1 to 26.   28. A method comprising administering the dosage form of claim 27 to a subject.   30. The method of claim 28, wherein the subject is in need of antigen specific tolerance.   30. The method of claim 28 or 29, wherein the dosage form is administered in an amount effective to produce a tolerogenic immune response specific for the APC presentable antigen.   31. The method of claims 28-30, wherein the dosage form is administered to the subject according to a protocol already known to produce a tolerogenic immune response specific for the APC presentable antigen in more than one subject. The method according to any one of the above.   32. The method of any one of claims 28-31, further comprising providing or identifying the subject.   33. The method of any one of claims 28-32, further comprising assessing the generation of a tolerogenic immune response specific for the APC presentable antigen in the subject.   34. Any of claims 28-33, wherein the subject has autoimmune disease, inflammatory disease, allergy, organ or tissue rejection, graft-versus-host disease, or has undergone or is scheduled to undergo a transplant. The method according to claim 1.   34. Any of the claims 28-33, wherein the subject has been administered, is being or will be administered a therapeutic protein that has caused, is causing or is expected to have an undesirable immune response. The method according to claim 1.   36. The method of any one of claims 28-35, wherein the dosage form is administered by intravenous, transmucosal, intraperitoneal, oral, subcutaneous, transpulmonary, intranasal, intradermal or intramuscular administration.   36. A method according to any one of claims 28 to 35, wherein the administration is performed by inhalation or intravenous, subcutaneous or transmucosal administration.   28. A composition according to any one of claims 1 to 26 or a dosage form according to claim 27 for use in therapy or prevention.   The subject who has or will be transplanted, or who has, is or will be administered a therapeutic protein that has caused, is or is expected to have an undesirable immune response 28. A composition according to any one of claims 1 to 26 or a dosage form according to claim 27 for use in the treatment or prevention of a subject who is   28. A composition according to any one of claims 1 to 26 or a dosage form according to claim 27 for use in a method according to any one of claims 28 to 37.   28. A composition according to any one of claims 1 to 26 or a dosage form according to claim 27 for use in a method for inducing a tolerogenic immune response.   27. A composition or claim according to any one of claims 1 to 26 for use in a method of prevention or treatment of autoimmune disease, inflammatory disease, allergy, graft-versus-host disease or unwanted immune response. 27. The dosage form according to 27.   28. A composition according to any one of claims 1 to 26 or a dosage form according to claim 27 for use in a method of treatment or prevention comprising administration by the route of claim 36 or claim 37. .   28. A composition according to any one of claims 1 to 26 or claim 27 for the manufacture of a medicament for use in a method according to any one of claims 28 to 37 and 41 to 43. Use of the described dosage form.